BIOLOGY 
UBffeRY 


BACTERIOLOGY 


GENERAL,   PATHOLOGICAL 


AND 


INTESTINAL 


BY 

ARTHUR  ISAAC  KENDALL,  B.S.,  PH.D.,  DR.P.H. 

PROFESSOR    OF    BACTERIOLOGY    IN    THE    NORTHWESTERN    UNIVERSITY    MEDICAL    SCHOOL, 

CHICAGO,  ILLINOIS 


ILLUSTRATED  WITH  98  ENGRAVINGS  AND  9    PLATES 


•XX 


LEA  &  FEBIGER 

PHILADELPHIA   AND  NEW   YORK 
1916 


BIOLOGY 
RA 


Entered  according  to  the  Act  of  Congress,  in  the  year  1916,  by 

LEA  &  FEBIGER, 
in  the  Office  of  the  Librarian  of  Congress.     All  rights  reserved. 


TO 

THEOBALD  SMITH,  M.D.,  LL.D. 


PREFACE. 


"!N  the  study  of  the  microscopic  forms  known  as  bacteria  we  have 
what  might  be  fitly  called  the  focal  points  of  the  various  branches  of 
biological  science.  Though  their  investigation  may  require  careful 
morphological  researches,  yet  the  unmistakable  monotony  of  form 
combined  with  a  considerable  variation  of  physiological  activity  has 
compelled  the  bacteriologist  to  pay  much  attention  to  means  by  which 
such  physiological  variations  may  be  more  or  less  accurately  registered 
in  order  that  they  may  serve  as  a  supplementary  basis  for  classification. 
Again,  with  unicellular  organisms  the  manifestations  of  cell  activity 
become  the  most  important  phenomena  for  study.  These  manifesta- 
tions bring  together  the  fields  of  physiology  and  chemistry  and  make 
bacteriology  in  one  sense  a  branch  of  physiological  chemistry."1 

"  There  is  no  ulterior  interest  in  the  study  of  bacteria  as  such,  which 
is  a  strong  impulse  in  many  other  departments  of  biological  science.  It 
is  what  bacteria  do,  rather  than  what  they  are,  that  commands  atten- 
tion, since  our  interest  centers  in  the  host  rather  than  the  parasite."2 

The  development  of  bacteriology  has  followed  very  closely  the 
gradual  improvement  of  the  optical  parts  of  the  compound  microscope, 
and  to  a  lesser  degree,  the  perfection  of  other  instruments  of  precision 
on  the  one  hand,  and  the  production  of  anilin  dyes  and  a  great  expan- 
sion of  the  fields  of  organic  and  physical  chemistry  on  the  other  hand. 
Naturally  the  greatest  advances  in  bacteriology  have  been  made  along 
the  lines  of  morphology,  staining  and  diagnosis,  because  the  application 
of  the  microscope,  anilin  dyes,  and  the  preparation  and  use  of  cultural 
media  to  bacterial  problems  is  relatively  simple  and  direct.  The  final 
chapters  of  bacteriology,  in  which  the  problems  of  immunology  are 
of  paramount  interest,  will  be  intimately  associated  with  an  unfolding 
of  the  chemistry  of  cellular  activity,  as  Theobald  Smith  has  so  clearly 
pointed  out  in  the  opening  paragraphs  of  this  discussion. 

1  Theobald  Smith.     The  Fermentation  Tube,  Wilder  Quarter  Century  Book,  1893, 
p.  187. 

2  Theobald  Smith.     Some  Problems  in  the  Life  History  of  Pathogenic  Microorgan- 
isms, Amer.  Med.,  19Q4,  viii,  711. 


vi  PREFACE 

The  chemistry  of  bacterial  activity  is  not  thoroughly  studied  at 
the  present  time  and  many  of  its  problems  must  await  the  develop- 
ment of  new  methods  of  chemistry  and  physics,  as  well  as  a  refine- 
ment of  existing  methods.  Nevertheless,  sufficient  information  exists 
to  warrant  its  presentation  in  concrete  form,  partly  to  emphasize  its 
deficiencies,  chiefly  to  indicate  its  relation  to  the  biology  of  the  bacteria, 
which  are  potentially  "living  chemical  reagents,"  as  Professor  Folin 
has  so  aptly  termed  them. 

In  the  last  analysis,  the  interest  and  importance  of  bacteria  centers 
around  "what  they  do  rather  than  what  they  are,"  and  the  elucida- 
tion of  this  aspect  of  bacteriology  lies  largely  within  the  field  of 
biochemistry. 

The  relation  of  the  chemistry  of  bacterial  nutrition  to  the  study 
of  intestinal  bacteriology  in  health  and  disease  is  self-evident;  some 
of  the  more  general  aspects  of  this  subject  are  briefly  set  forth  in  the 
chapter  relating  to  intestinal  bacteria. 

It  is  with  great  pleasure  that  the  writer  acknowledges  his  indebted- 
ness to  his  colleagues  in  the  Northwestern  Univeristy  Medical  School 
for  many  valuable  suggestions,  to  Doctors  Noguchi  and  -Amoss,  of 
the  Rockefeller  Institute,  for  the  privilege  of  using  the  original  plates 
illustrating  the  Treponemata  and  Poliomyelitis,  and  to  Mrs.  N.  M. 
Frain  for  the  line  drawings  in  the  text.  Finally,  the  writer  would 
acknowledge  his  deep  obligation  to  Miss  Bertha  J.  Schwarz,  Secretary 
of  the  Department  of  Bacteriology,  for  her  invaluable  assistance  in 
the  preparation  of  the  manuscript  and  in  reading  the  proof  of  the 
book.  A.  I.  K. 

CHICAGO,  1916. 


CONTENTS. 


SECTION  I.-GENERAL  BACTERIOLOGY. 

INTRODUCTION.— THE  DEVELOPMENT  AND  SCOPE  OF 
BACTERIOLOGY. 

CHAPTER  I. 
THE  MORPHOLOGY  OF  BACTERIA. 

PAGE 

Normal  and  Abnormal  Forms — Size  and  Weight — Structure  and  Con- 
stituents of  the  Bacterial  Cell— Reproduction  and  Cell  Division — 
Cell  Grouping,  Classification,  and  Mutation 17-35 

CHAPTER  II. 

THE  PHYSIOLOGY  OF  BACTERIA  AND  THE  EFFECT  OF  ENVIRONMENTAL 

INFLUENCES. 

Rate  of  Reproduction — Motility — Germination  of  Spores — Longevity — 
Effects  of  Moisture,  Oxygen,  Temperature,  Light  and  Electricity, 
Gravity,  Osmotic  Pressure — Production  of  Enzymes, "Toxins,  Pto- 
maines and  Pigments — Symbiosis,  Antibiosis,  Commensalism  .  .  36-55 


CHAPTER  III. 
THE  CHEMISTRY  OF  BACTERIA. 

Chemical  Constitution  of  Bacteria  and  Composition  of  Morphological 
Components  of  Bacterial  Cell — Food  Relationships  of  Bacteria, 
Bacterial  Nutrition 56-67 

CHAPTER  IV. 
BACTERIAL  METABOLISM. 

The  Nature  of  Bacterial  Metabolism — Nitrogen  Metabolism,  Carbon 
Metabolism — Reactions  of  Bacterial  Metabolism — Significance  of 
Bacterial  Metabolism — Putrefaction  and  Fermentation  .  68-83 


viii  CONTENTS 

CHAPTER  V. 
SAPROPHYTISM,  PARASITISM  AND  PATHOGENISM. 

PAGE 

The  Cycle  of  Parasitism — The  Cycle  of  Pathogenism — Distribution  of 
Parasitic  and  Pathogenic  Bacteria  in  Nature — How  Parasitic  and 
Pathogenic  Bacteria  Reach  Man — How  they  Reach  the  Body, 
Portals  of  Entry,  Where  They  Multiply  in  the  Body,  Where  and 
How  They  Escape  from  the  Body — Balanced  Pathogenism  and 
Epidemiology 84-110 

CHAPTER  VI. 

INFECTION  AND  IMMUNITY. 

Classification  of  Immunity — Infection — Theories  of  Immunity     .      .      .     111-131 

CHAPTER  VII. 
Anaphylaxis,  Allergy  or  Hypersensitiveness 132-141 

CHAPTER  VIII. 
ANTIGENS  AND  THE  TECHNIQUE  OF  SERUM  REACTIONS. 

Nature  of  Antigens  and  Antibodies — Agglutinins  and  Precipitins — 
Lysins,  Hemolysis  and  Complement  Fixation — Aggressins — Opson- 
ins,  Tropins — Bacterial  Vaccines  .  ....."..  .  .  142-174 

CHAPTER  IX. 
BACTERIOLOGICAL  TECHNIQUE. 

Methods  for  the  Microscopic  Study  of  Bacteria — Staining  Methods- 
Media — Cultivation  of  Bacteria,  Study  of  Bacterial  Cultures  .  .  175-223 

CHAPTER  X. 
BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL  FROM  PATIENT  AND  CADAVER. 

Autopsy  Procedure — Blood  Cultures — Cerebrospinal  Fluid — Peritoneal, 
Pleural  and  Pericardial  Fluids— Pus— Examination  of  Urine, 
Feces,  Sputum,  Buccal  and  Pharyngeal  Material— Bacteriological 
Examination  of  the  Eye,  Ear  and  Nose— The  Utilization  of  Animals 
for  Bacterial  Diagnosis  and  Experimentation  .......  224-240 

CHAPTER  XI. 
PRACTICAL  STERILIZATION,  ANTISEPSIS  AND  DISINFECTION. 

Laboratory  Sterilization— Physical  Agents,  Chemical  Solutions,  Test- 
ing and  Standardizing  Liquid  Disinfectants— Gaseous  Disinfectants 
—Disinfection  of  Sputum,  Vomitus,  Feces  and  Urine,  Fomites,  Skin 
and  Hands,  Instruments  .  .  ...  -V  241-254 


CONTENTS  IX 

SECTION  II.-PATHOGENIC  BACTERIA. 

CHAPTER  XII. 

PAGE 

The  Pyogenic  Cocci 255-268 

CHAPTER  XIII. 
The  Streptococcus-Pneumococcus  Group 269-291 

CHAPTER  XIV. 
The  Meningococcus-Gonococcus  Group 292-309 

CHAPTER  XV. 
Micrococcus  Melitensis 310-312 

CHAPTER  XVI. 
The  Alcaligenes — Dysentery — Typhoid — Paratyphoid  Group       .      .      .     313-352 

CHAPTER  XVII. 
The  Coli— Cloacae— Proteus  Group 353-362 

CHAPTER  XVIII. 
The  Mucosus  Capsulatus  Group 363-366 

CHAPTER   XIX. 

Glanders,  Anthrax,  Pyocyaneus,  Infectious  Abortion,  Aciduric  Bacteria      367-387 

CHAPTER  XX. 

Diphtheria  Group •  .      .      .     388-406 

CHAPTER  XXI. 
Hemorrhagic  Septicemia  Group 407-416 

CHAPTER   XXII. 

HEMOGLOBINOPHILIC  BACILLI. 
Influenza,  Pertussis,  Koch- Weeks,  Morax-Axenfeld  and  Ducrey  Bacilli  .     417-427 

CHAPTER  XXIII. 

TUBERCLE  BACILLUS  GROUP. 
Human,  Bovine  and  Avian ...     428-462 


x  CONTENTS 

CHAPTER  XXIV. 

PAGE 

Leprosy  and  Acid-fast  Bacteria  other  than  the  Tubercle  Group  .  463-471 

CHAPTER  XXV. 

ANAEROBIC    BACTERIA. 

Tetanus,   Botulinus,   Aerogenes    Capsulatus,   Malignant    Edema   and 

Symptomatic  Anthrax .      .     472-498 

CHAPTER  XXVI. 
Cholera  Group 499-513 

CHAPTER  XXVII. 
Treponemata  and  Spirocheta 514-532 


SECTION  III.-HIGHER  BACTERIA,  MOLDS, 
YEASTS,  FILTERABLE  VIRUSES,  AND 
DISEASES  OF  UNKNOWN  ETIOLOGY. 

CHAPTER  XXVIII. 

Trichomycetes,  Actinomycetes,  Hyphomycetes  and  Saccharomycetes     .      533-554 

CHAPTER  XXIX. 
Filterable  Viruses — Diseases  of  Unknown  Etiology 555-578 

SECTION  IV.-GASTRO-INTESTINAL 
BACTERIOLOGY. 

CHAPTER  XXX. 
Gastro-intestinal  Bacteriology 579-600 

SECTION  V.-APPLIED  BACTERIOLOGY. 

CHAPTER  XXXI. 
Bacteriology  of  Milk 601-613 

CHAPTER  XXXII. 

Bacteriology  of  the  Soil,  Water  and  Air '^     .     .     .      .     614-625 


SECTION    I. 

GENERAL  BACTERIOLOGY. 


INTRODUCTION— THE    DEVELOPMENT   AND   SCOPE    OF 

BACTERIOLOGY. 

BACTERIOLOGY  is  that  branch  of  Natural  Science  which  treats  of 
the  structure,  functions  and  chemistry  of  bacteria.  Bacteria  are 
intimately  related  to  many  fields  of  human  activity,  therefore  bacterio- 
logy is  inseparably  associated  with  a  number  of  the  arts  and  sciences. 
In  those  branches  of  science  which  treat  of  the  diseases  of  plants, 
of  animals  and  of  man,  bacteria  enter  into  complex  reciprocal  relations 
with  their  hosts  as  parasites  or  pathogens,  relations  which  are  neither 
purely  bacterial,  animal  nor  vegetal  in  their  limitation.  A  new  science, 
Immunology,  is  rapidly  developing  which  is  concerned  chiefly  with 
the  elucidation  of  these  relationships  between  host  and  parasite. 

Bacteria  are  the  smallest  in  size  and  simplest  in  structure  of  known 
visible  living  organisms.  They  are  rigid  unicellular  organisms  devoid 
of  chlorophyll  or  other  photodynamic  pigment;  they  possess  no 
morphologically  demonstrable  nucleus  and  reproduce  by  simple 
transverse  fission,  the  resulting  individuals  being  of  approximately 
equal  size. 

Bacteria  are  ubiquitous  in  their  distribution;  they  are  found  in  all 
climates  in  association  with  animal  and  vegetable  life.  Some  thrive 
at  temperatures  but  slightly  above  the  freezing  point  of  water;  the 
majority  flourish  between  15°  and  40°  Centigrade;  some  even  develop 
in  thermal  springs  at  a  temperature  of  70°  Centigrade.  Free  or  atmos- 
pheric oxygen  is  essential  for  most  types  of  bacteria,  but  to  a  few  it 
is  actually  a  poison. 

Bacteria  are  ordinarily  classed  as  plants,  but  they  exhibit  several 
prominent  characteristics  which  suggest  a  relationship  with  the  lowest 

animals.    The  most  important  of  these  is  the  absence  of  photodynamic 
2 


18        /.  ','•  ".        ;  •.  :  INTRODUCTION 

pigment   (chlorophyll),  which  implies  an  analytical  or  destructive 
function  in  the  economy  of  Nature. 

The  great  majority  of  bacteria  are  saprophytic,  living  upon  dead 
organic  matter,  which  they  transform  into  simple  compounds  suitable 
for  plant  use.  These  bacteria  are  Nature's  analysts.  Some  are  para- 
sitic on  living  plants  and  animals;  a  few  are  progressively  pathogenic 
for  man  and  animals.  It  is  this  last  group,  few  in  numbers,  but  for- 
midable in  that  their  activities  are  in  partial  opposition  to  those  of 
man  and  animals,  that  has  given  to  bacteria  all  the  notoriety  which 
they  possess. 

Anton  von  Leeuwenhoek,  a  Dutch  spectacle  maker,  appears  to  have 
been  the  first  to  see  bacteria:  in  1675,  with  lenses  of  his  own  grinding, 
he  examined  various  putrescent  fluids,  drops  of  water,  scrapings  from 
his  teeth,  and  his  own  diarrheal  discharges.  He  says  in  his  writings, 
collected  and  edited  by  Robert  Hooke,1  "With  great  astonishment 
I  observed  everywhere  through  the  material  which  I  was  examining, 
animalcules  of  the  most  minute  size,  which  moved  themselves  about 
very  energetically."  It  is  possible  to  recognize  cocci,  bacilli  and 
spirilla  in  his  drawings,  and  it  is  almost  certain  that  he  actually  observed 
motility  among  his  organisms.  The  learned  monk,  Athanasius  Kircher, 
observed  and  described  "minute  living  worms"  as  early  as  1659,  but 
his  optical  equipment  was  inferior  to  that  of  von  Leeuwenhoek  and  it 
is  doubtful  if  he  actually  saw  bacteria. 

Improvements  in  the  microscope  opened  a  new  world  for  investiga- 
tion and  speculations  concerning  the  doctrine  of  the  Spontaneous 
Generation  of  Life  led  to  numerous  experiments  of  increasing  refine- 
ment that  finally  resulted  in  the  brilliant  researches  of  Pasteur,  and 
Tyndall,  who  showed  by  numerous  ingenious  and  carefully  executed 
experiments  that  the  phenomena  in  putrescible  fluids  erroneously 
interpreted  as  spontaneous  generation  did  not  take  place  when  proper 
precautions  in  manipulation  were  observed.  About  1835  achromatic 
lenses  for  the  microscope  reached  a  state  of  perfection  compatible 
with  the  examination  of  minute  objects  and  the  microscope  was  almost 
immediately  applied  to  the  study  of  various  morbid  processes,  with 
remarkable  success.  Bassi  (1837)  discovered  a  fungus  which  caused 
a  contagious  disease  of  silk  worms  known  as  muscardine;  Cagniard 
de  Latour  and  Schwann  observed  and  described  the  yeast  plant  in 
liquids  undergoing  alcoholic  fermentation. 

1  Collected  Memoirs  of  Anton  v.  Leeuwenhoek,  Royal  Society  of  London,  1675,  1683. 


INTRODUCTION  19 

Ehrenberg  (1838)  began  his  classification  of  animalcules  and  in  his 
group  of  Vibrionia  described  several  "species"  of  organisms,  as  follows: 

1.  Bacterium — rigid  and  filamentous  organisms. 

2.  Vibrio — flexuous  and  filamentous  organisms. 

3.  Spirillum — rigid  spiral  filamentous  organisms. 

4.  Spirocheta — flexuous  spiral  filamentous  organisms. 

This  classification,  which  contains  terms  widely  used  in  bacterial 
nomenclature  today,  was  followed  in  1872  by  the  important  contribu- 
tions of  Cohn  upon  "Bacteria,"  the  starting-point  of  modern  bacterial 
classification. 

The  diseases  of  man  naturally  attracted  much  attention  and  in  1839 
Schoenlein  examined  the  crusts  of  that  disease  of  the  scalp  known  as 
favus  with  the  microscope  and  found  the  mycelia  of  the  fungus  now 
known  in  his  honor  as  Achorion  schoenleinii. 

The  extensive  studies  of  Pasteur  upon  yeasts  and  the  "diseases" 
of  beer  and  wine,  upon  the  diseases  of  the  silk  worm  (pebrine  and 
flacherie),  upon  furunculosis  and  puerperal  sepsis,1  upon  anthrax 
and  anthrax  immunization  (attenuated  viruses)  chicken  cholera,  and 
somewhat  later,  rabies  laid  broad  foundations  for  the  development 
of  the  science  of  bacteriology. 

Among  the  most  important  technical  discoveries  which  have  con- 
tributed to  the  development  of  bacteriology  are:  The  improvement 
in  the  achromatic  lens  (about  1835)  and  the  perfection  of  the  sub- 
stage  condenser  (Abbe) ;  the  use  of  cotton  for  air  filters  in  flasks  and 
test-tubes  by  Schroeder  and  von  Dusch  (1854),  the  sterilization  of 
culture  media  by  heat  (Pasteur,  Tyndall,  Koch  and  others),  the 
introduction  of  anilin  dyes  as  staining  reagents  by  Weigert  and  Ehrlich 
(1877),  and  finally,  the  use  of  solid  culture  media  and  the  plate  method 
for  pure  cultures  by  Koch  in  1881. 

Sir  Joseph  Lister  (1867)  published  an  epoch-making  contribution 
entitled,  "On  the  Antiseptic  Principle  of  the  Practice  of  Surgery," 
in  which  is  clearly  set  forth  the  importance  of  bacteria  in  surgery 
and  the  principles  of  surgical  asepsis  that  have  revolutionized  this 
branch  of  medicine. 

About  1878  Koch  isolated  the  anthrax  bacillus  in  pure  culture  from 
the  blood  of  infected  animals,  grew  the  organisms  for  several  generations 
in  the  clear  aqueous  humor  of  the  eye  of  the  ox,  and  then  reinjected 
the  organisms  into  experimental  animals  and  reproduced  the  disease. 
For  the  first  time  a  specific  microbe  was  clearly  and  convincingly 

1  Compt.  rend.  Acad.  d.  Sci.,  1880,  xc,  1033. 


20  INTRODUCTION  . 

shown  to  be  the  etiological  factor  of  a  bacterial  disease.  Koch  also 
found  that  the  anthrax  bacillus  formed  spores. 

From  this  time  bacteriology  developed  with  amazing  rapidity.  In 
1882  Koch  startled  the  world  with  the  announcement  of  the  dis- 
covery of  the  tubercle  bacillus;  and  in  rapid  succession,  typhoid, 
diphtheria,  cholera,  tetanus  and  other  well-known  pathogenic  bacteria 
were  isolated  and  studied  in  pure  culture. 

In  1882  Metchnikoff  published  the  first  of  his  highly  important 
contributions  to  immunity  and  phagocytosis,  and  a  decade  later  von 
Behring  and  Kitasato  announced  the  discovery  of  diphtheria  antitoxin. 

The  last  three  decades  have  not  only  witnessed  the  rise  and  develop- 
ment of  those  most  brilliant  chapters  of  medicine,  infection  and  im- 
munity; but  sanitation,  agriculture,  many  industries  and  other  fields 
of  human  activity  have  benefited  largely  by  the  development  of 
bacteriology. 

In  medicine  the  diagnosis  of  bacterial  disease  has  reached  a  high 
degree  of  precision,  and  bacteriological  diagnosis  is  an  important 
branch  of  medical  science.  The  most  important  problem  for  the  future 
is  to  create  a  system  of  Bacterial  Therapeutics  of  equal  efficiency. 


CHAPTER  I. 


THE  MORPHOLOGY  OF  BACTERIA. 


A.  MORPHOLOGY — NORMAL      FORMS: 

Coccus,  BACILLUS,  SPIRILLUM. 

B.  MORPHOLOGY — ATYPICAL   AND   AB- 

NORMAL FORMS. 

1.  Variation. 

2.  Degeneration    and    Involution. 

3.  Pleiomorphism. 

4.  Branching. 

C.  SIZE    OF    BACTERIA:    WEIGHT    OF 

BACTERIA. 

D.  STRUCTURE   AND   CONSTITUENTS   OF 

THE  BACTERIAL  CELL. 
1.  Cell     Membrane,     Ectoplasm, 
Capsule,  Zooglea. 


2.  Cell  Substance,  Cytoplasm, 
Nucleus,  Metachromatic  and 
Polar  Granules,  Flagella, 
Spores,  Germination  of  Spores, 
Arthrospores. 

E.  REPRODUCTION  AND  CELL  DIVISION 

IN  BACTERIA. 

F.  CELL  GROUPING. 

G.  CLASSIFICATION  OF  BACTERIA. 

1.  Relation  of  Bacteria  to  Higher 

Plants. 

2.  Classification. 

H.  MUTATION.     CONSTANCY  OF  TYPES. 


A.     NORMAL  FORMS:  COCCI,  BACILLI,  SPIRILLA. 

THE  normal  forms  of  the  true  bacteria  are  very  simple,  and  are 
included  in  three  fundamental  types:  the  sphere  (coccus,  plural  cocci), 
the  straight  rod  (bacillus,  plural  bacilli),  and  the  curved  rod  (spirillum, 
plural  spirilla) .  There  is  in  addition  a  group  of  organisms  intermediate 
between  the  true  bacteria  and  the  molds,  which  is  characterized  by 
a  filamentous  type  of  growth.  The  members  comprising  this  group 
of  filamentous  organisms  are  commonly  known  as  the  higher  bacteria 
or  Chlamydobacteriacese.  An  organism  belonging  to  one  of  these 
groups  always  reproduces  its  kind  under  normal  conditions;  that  is, 
a  coccus  always  reproduces  a  coccus,  a  bacillus  always  reproduces  a 
bacillus,  and  a  spirillum  always  reproduces  a  spirillum. 

Cocci. — A  single  coccus  is  typically  spherical,  although  those 
organisms  in  which  division  is  taking  place  may  be  temporarily  some- 
what elongated  in  one  diameter,  thus  appearing  oval  in  outline  at  this 
stage  of  their  development.  They  may  even  resemble  very  short 
bacilli  in  extreme  instances.  The  habitual  occurrence  of  cocci  in  pairs, 
frequently  with  their  proximate  surfaces  flattened,  is  a  noteworthy 
morphological  characteristic  of  certain  members  of  this  group.  They 
are  referred  to  as  diplococci.  The  flattening  of  the  proximated  sur- 
faces may  be  associated  with  an  elongation  of  the  axes  of  the  organisms 
parallel  to  the  plane  of  apposition,  which  leads  to  "coffee  bean" 


22  THE  MORPHOLOGY  OF  BACTERIA 

shaped  diplococci,  exemplified  in  the  meningococcus  and  gonococcus, 
or  to  an  elongation  of  the  axes  perpendicular  to  the  plane  of  apposition, 
in  which  event  the  organisms  are  "lance-shaped"  diplococci,  as  for 
example  the  pneumococcus. 

Bacilli. — Bacilli  are  rod-shaped,  cylindrical  organisms  in  which  a 
longer  and  a  shorter  dimension  may  be  recognized.  They  are  typi- 
cally circular  in  cross-section.  When  division  is  taking  place  the  shorter 
bacilli  may  be  temporarily  oval  or  even  circular  in  outline.  The  dimen- 
sions of  bacilli  vary  considerably:  some  are  habitually  long,  some  are 
short,  some  are  thick,  some  are  thin.  The  ends  may  be  convex,  less 
commonly  flat  or  even  concave.  A  few  bacilli  are  not  typically  isodia- 
metric,  but  appear  in  outline  as  club-shaped,  spindle-shaped,  or  even 
more  or  less  conical  (cuneate)  rods.  Less  commonly,  slightly  curved 
rods  are  met  with;  the  curvature  takes  place  along  the  longer  dimension. 


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FIG.  1. — The  normal  types  of  bacteria.  1-6,  cocci;  7-13,  bacilli;  14-16,  spirilla; 
1,  micrococcus;  2  and  3,  diplococci;  4,  tetracoccus;  5,  sarcina;  6,  streptococcus  (the 
lower  chain  includes  an  arthrospore) ;  7  and  8,  bacilli;  9,  10,  12,  and  13,  bacilli  with 
various  granules;  11,  strep tobacillus ;  14,  vibrio;  15,  spirillum;  16,  Spirocheta  trepo- 


Spirilla. — Spiral  bacteria,  like  the  bacilli,  exhibit  a  longer  and 
a  shorter  dimension;  unlike  the  bacilli,  the  longer  axis  is  curved  in 
three  planes  of  space.  The  curvature  may  be  slight,  less  than  a  com- 
plete turn,  in  which  event  the  organism  is  "comma-shaped"  when 
viewed  under  the  microscope;  it  may  be  a  series  of  open  curves,  giving 
the  organism  a  sinuous  outline;  or  it  may  be  very  much  curved,  so 
that  the  organism  resembles  a  somewhat  closely  coiled  spring  in  out- 
line. As  a  rule,  the  curvature  is  symmetrical  and  uniform  in  each 
instance. 

The  cocci,  through  almost  imperceptible  morphological  gradations, 
merge  into  the  bacilli,  and  the  bacilli,  through  the  slightly  curved 
forms,  merge  into  the  spirilla.  Even  in  the  spirilla  slight  differences 
in  curvature  are  usually  discernible.  Thus,  a  culture  of  the  cholera 
vibrio  may  contain  many  straight,  uncurved  organisms  in  addition 


ABNORMAL  FORMS  23 

to  the  slightly  curved  rods  which  are  the  characteristic  morphologic 
forms.  There  are  a  few  bacteria  in  which  the  morphology  is  still  a 
subject  of  controversy.  For  example,  Micrococcus  melitensis  is  called 
Bacillus  melitensis  by  some  observers.  The  vast  majority  of  bacteria, 
however,  are  easily  referable  to  their  proper  morphological  type  by 
simple  inspection  under  the  microscope. 

B.     ABNORMAL  FORMS:  VARIATION,   DEGENERATION  AND  IN- 
VOLUTION, PLEIOMORPHISM  AND  BRANCHING. 

Variation. — The  composition  of  the  medium  in  which  bacteria  are 
growing,  the  age  of  the  culture,  and  to  a  limited  degree  even  the  tem- 
perature of  incubation  influence  somewhat  the  average  size  of  bacteria. 
Given  constant  conditions,  however,  bacteria  growing  in  a  favorable 
environment  exhibit  constancy  of  form  and  size,  although  a  few  organ- 
isms in  every  culture  are  somewhat  larger  or  smaller  than  their  fellows, 
appearing  as  occasional  giants  or  dwarfs.  These  occasional  giant  and 
dwarf  forms  represent  normal  variations  in  size  from  the  average  or 
mean. 

Degeneration  and  Involution. — Bacteria  growing  in  an  unfavorable 
environment,  brought  about  by  the  accumulation  of  waste  products, 
by  undue  changes  in  reaction  resulting  in  excessive  acidity  or  alka- 
linity, by  the  presence  of  harmful  chemicals,  or  by  specific  antago- 
nistic substances,  may  gradually  assume  atypical  shapes,  probably  the 
direct  result  of  these  harmful  influences.  These  atypical  organisms 
may  exhibit  little  or  no  resemblance  to  the  normal  organism,  either  in 
form  or  size;  they  may  or  may  not  develop  into  normal  organisms  when 
they  are  placed  again  in  a  favorable  environment.  If  the  change  is 
a  morphological  one,  the  atypical  organisms  are  designated  involution 
forms :  thus,  plague  bacilli  grown  in  nutrient  agar  containing  3  per  cent, 
common  salt  appear  as  swollen,  balloon-like  bodies,  notably  unlike 
the  typical  short  rod-shaped  bacillus.  If,  on  the  contrary,  the 
organisms  permanently  lose  some  morphological  or  chemical  charac- 
teristic, they  are  spoken  of  as  degeneration  forms.  Thus,  anthrax 
bacilli  heated  for  several  hours  at  43°  to  44°  C.  lose  their  ability  to 
form  mature  spores. 

Pleiomorphism. — By  pleiomorphism  is  meant  a  permanent  or  semi- 
permanent change  in  the  normal  form  of  the  organism.  A  pleio- 
morphic  organism  would  be  one  which  might  at  one  time  resemble 
a  bacillus,  again  a  coccus,  or  even  a  spirillum,  depending  upon  the  age 


24  THE  MORPHOLOGY  OF  BACTERIA 

and  growth  of  the  organism  or  the  fitness  of  the  culture  medium.  This 
phenomenon  is  rarely  or  never  met  with  among  the  pathogenic  bacteria. 

Branching. — Among  the  individual  organisms  comprising  a  culture 
in  artificial  media  of  tubercle,  diphtheria  or  glanders  bacilli,  and  to  a 
lesser  extent  of  other  bacilli,  a  certain  number  appear  as  definitely 
branched  rods:  the  typical  organism  in  each  instance  does  not  exhibit 
branching.  Branching  has  also  been  demonstrated  in  the  spirilla.1 
Bacillus  bifidus  appears  habitually  as  a  rod-shaped  organism  with 
bifurcated  ends  in  artificial  media,  although  it  is  an  unbranched 
bacillus  in  its  normal  habitat,  the  intestinal  tract  of  nurslings.  Occa- 
sionally, bacteria,  as  the  tubercle  bacillus,  may  exhibit  branching  in 
the  animal  body  as  well  as  in  cultures,  although  less  commonly. 

The  cause  of  this  branching  is  unknown,  and  at  least  two  theories 
have  been  advanced  in  explanation  of  it:  each  theory  has  a  certain 
amount  of  evidence  in  its  favor.  One  theory  assumes  that  branching 
is  the  result  of  unfavorable  environmental  conditions,  and  it  has  been 
shown  that  old  broth  cultures  of  diphtheria  bacilli  contain  branched 
organisms;  young  cultures  contain  few  or  no  branched  forms.  The 
assumption  is  that  old  cultures  contain  harmful  products  of  metab- 
olism which  cause  the  diphtheria  bacillus  to  assume  branched  forms. 
The  second  theory  asserts  that  the  appearance  of  branched  forms 
among  bacteria  demonstrates  a  relationship  between  them  and  higher 
organisms,  which  are  habitually  branched.  Bacteria,  according  to 
this  theory,  exhibit  branching  as  a  part  of  their  normal  development. 

Branching  does  not  necessarily  take  place  under  conditions  which 
would  appear  to  be  unfavorable  or  partially  inimical  to  their  growth, 
and,  on  the  other  hand,  it  may  be  observed  occasionally  when  environ- 
mental conditions  should  be  favorable  for  development.  It  appears 
to  be  reasonable  to  assume  that  branching  may  be  a  normal  develop- 
mental process  in  the  life  history  of  the  organism,  although  the  phylo- 
genetic  significance  of  branching  is  as  yet  undetermined. 

C.     SIZE   AND   WEIGHT   OF   BACTERIA. 

Size. — The  unit  of  measurement  for  microscopic  objects  is  the 
micron  (/*),  which  is  0.001  of  a  millimeter,  or  approximately  ^m  of 
an  inch,  in  length.  Bacteria  are  the  smallest  known  living  organisms 
which  have  been  seen  with  the  microscope.  Measured  with  this  unit, 
they  exhibit  considerable  differences  in  size.  The  average  sized  pus- 

1  Reichenbach,  Centralbl.  f.  Bakteriol.,  1901,  xxix  ,553. 


STRUCTURE  AND  CONSTITUENTS  OF  BACTERIAL  CELL      25 

producing  coccus  is  0.8  micron  in  diameter;  Micrococcus  melitensis, 
the  smallest  of  the  Coccacese,  varies  in  diameter  from  0.3  to  0.5  micron. 
The  largest  known  bacillus,  B.  biitschlii1  is  3  to  6  microns  in  diameter 
and  from  40  to  60  microns  in  length.  The  smallest  known  bacillus, 
B.  influenzse,  is  but  0.2  by  0.5  micron  in  diameter;  an  average  sized 
bacillus  would  measure  about  2  microns  in  length  and  1  micron  in 
diameter.  Spirillum  colossum2  is  from  2.5  to  3.5  microns  in  diameter. 
The  cholera  vibrio  is  about  2.5  microns  long  and  1  micron  in  diameter. 
There  are  certain  living  viruses  of  unknown  morphology,  so-called 
ultramicroscopic  or  filtrable  viruses,  which  are  either  somewhat 
smaller  than  any  known  bacteria  or  more  plastic.  Viruses  belonging 
to  this  group  derive  their  name  from  the  fact  that  they  retain  their 
viability  even  after  passage  through  the  pores  of  standard,  unglazed 
porcelain  filters,  which  will  hold  back  even  the  smallest  bacteria. 

Weight  of  Bacterial  Cell. — The  weight  of  a  bacterial  cell  is  depend- 
ent upon  its  size  and  its  specific  gravity.  According  to  Rubner,3  the 
specific  gravity  of  common  bacteria  varies  between  1.038  and  1.065.4 
B.  coli  is  an  average  sized  cylindrical  rod  (bacillus),  measuring  1 
micron  in  diameter  and  2  microns  in  length.  The  volume  of  a  cylinder 
is  the  product  of  the  diameter  squared,  multiplied  by  0.7854,  multiplied 
by  the  length  of  the  cylinder.  The  volume  of  a  single  colon  bacillus 
consequently  would  be  (0.001)2  X  0.7854  X  0.002,  or  0.00000000157 
c.mm.  The  weight  of  a  single  colon  bacillus  would  be  the  volume 
multiplied  by  the  specific  gravity,  which  is  approximately  1.040  or 
0.00000000163  mg.;  that  is  to  say,  sixteen  hundred  million  colon  bacilli 
would  weight  approximately  one  milligram.  For  purposes  of  com- 
parison it  may  be  stated  that  a  single  red  blood  corpuscle  (human) 
weighs  about  0.00008  mg.,  about  fifty  thousand  times  the  weight 
of  a  single  colon  bacillus. 

D.     STRUCTURE  AND  CONSTITUENTS  OF  THE  BACTERIAL  CELL. 

The  typical  bacterial  cell  consists  essentially  of  protoplasmic  cell 
substance,  endoplasm,  enclosed  by  a  rigid  cell  membrane,  ectoplasm. 

1.  Cell  Membrane. — Ectoplasm. — Bacteria  appear  to  possess  a 
special  external  boundary  layer,  cell  membrane,  or  ectoplasm,  which  is 

1  Schaudinn,  Arch.  f.  Protistenk.,  1902,  i,  306. 

2  Errera,  Recueil  de  1'Instit.  botanique  (Universite  de  Bruxelles),  1901,  v,  347. 

3  Arch.  f.  Hyg.,  1903,  xlvi,  41;  1890,  xi,  385. 

4  Stigell  (Cent.  f.  Bakt.,  1908,  xlv,  487)  finds  that  the  specific  gravity  of  the  same 
organism  varies  somewhat  with  the  medium  in  which  it  is  grown.     The  specific  gravity 
of  ordinary  bacteria  varies  commonly  between  1.120  and  1.35,  older  cultures  being  as 
a  rule  of  less  specific  gravity  than  younger  cultures  of  the  same  kind. 


26  THE  MORPHOLOGY  OF  BACTERIA 

rigid  and  maintains  the  shape  of  the  organism.  Generally  speaking, 
this  cell  membrane  is  intermediate  in  character  between  that  char- 
acteristic of  animal  and  of  plant  cells  respectively,  being  somewhat 
more  developed  than  the  former,  less  highly  specialized  as  a  rule  than 
the  latter.  Some  authorities  consider  the  cell  membrane  of  bacteria 
to  be  merely  a  concentrated  external  layer  of  endoplasm. 

The  thickness  of  the  cell  membrane  varies  among  different  varieties 
of  bacteria,  and  it  appears  to  be  somewhat -thinner  in  young  organisms 
of  a  given  variety  than  in  the  older  individuals  of  the  same  kind. 
Ordinarily  it  is  not  seen,  and  special  stains  are  required  to  demonstrate 
it  clearly.  In  certain  spore-forming  bacteria,  however,  the  cell  mem- 
brane is  occasionally  seen  after  the  spore  has  matured  within  the  cell, 
as  a  thin,  feebly  staining  shadow,  outlining  the  original  contour  of  the 
organism.  Bacteria  which  plasmolyze  easily  also  show  the  cell  wall 
clearly  after  the  cell  contents  have  shrunken  away  from  it. 

Capsule. — A  considerable  number  of  bacteria  are  surrounded  by 
mucin-like  envelopes,  particularly  when  they  are  observed  in  the  animal 
body  or  grown  in  albuminous  fluids.  This  envelope  or  capsule  fre- 
quently disappears  when  the  organisms  are  grown  in  ordinary  media. 
This  has  led  to  the  theory  that  a  capsule  represents  an  hypertrophy 
of  the  ectoplasm.  The  significance  of  capsules  is  still  a  matter  of 
controversy.  Two  principal  theories  have  been  advanced  to  explain 
the  significance  of  capsules :  according  to  one  theory,  bacterial  capsules 
are  purely  degenerative  phenomena;  the  more  widely  accepted  theory, 
which  has  much  evidence  in  its  favor,  maintains  that  capsule  formation 
is  closely  related  to  the  virulence  of  the  organisms.1  The  demonstration 
of  capsules  may  be  an  important  factor  in  the  identification  of  certain 
bacteria,  for  example,  the  pneumococcus. 

Zooglea. — A  very  few  bacteria  exhibit  a  slimy  intracellular  substance 
which  causes  cohesion  between  considerable  numbers  of  bacterial 
cells.  This  intracellular  substance,  zooglea,  is  colored  lightly  by 
ordinary  staining  methods.  It  is  not  found  in  any  of  the  pathogenic 
bacteria. 

2.  Cell  Substance. — Cytoplasm. — The  cytoplasm  or  endoplasm  of 
living  bacteria  (particularly  in  young  cultures)  is  usually  a  clear, 
colorless,  highly  refractile,  homogeneous  appearing  substance,  although 
at  times  various  granules  may  be  seen  within  it.  Vacuoles  also  are 
met  with,  usually  in  older  bacteria.  The  cytoplasm  usually  stains 
readily  with  basic  anilin  dyes.  A  few  bacteria,  notably  B.  viride  and 

1  Eisenberg,  Centrabl.  f.  Bakteriol.,  1908,  xlv,  148. 


STRUCTURE  AND  CONSTITUENTS  OF  BACTERIAL  CELL      27 

B.  chlorinum,  contain  a  yellowish  pigment  in  the  cytoplasm  suggesting 
chlorophyll,  and  the  so-called  purple  bacteria  similarly  possess  a 
purple  colored  pigment,  bacteriopurpurin. 

Nucleus. — The  occurrence  of  a  demonstrable  morphological  nucleus 
in  bacteria  is  by  no  means  definitely  settled:  the  typical  bacterial 
cell  can  not  be  separated  chromoscopically  into  a  nucleus  and  cyto- 
plasm. Those  who  have  thoroughly  studied  the  question  by  staining 
methods,  notably  Nakanishi,1  believe  that  the  whole  bacterial  cell, 
as  it  is  ordinarily  seen,  is  potentially  a  nucleus  surrounded  by  a  very 
thin  film  of  cytoplasm.  Others  believe  the  nucleus  substance  is  dis- 
tributed throughout  the  cell  in  very  finely  divided  granules :  Zettnow2 
is  the  champion  of  the  latter  theory.  He  believes  that  the  bacterial 
cell,  as  it  is  viewed  following  the  usual  staining  processes,  is  endoplasm 
in  which  the  nuclear  substance  is  finely  divided  and  uniformly  dis- 
tributed. Some  observers  deny  that  a  nucleus  exists  at  all.  Chemical 
analyses  show  beyond  doubt  that  bacteria  contain  a  relatively  high 
percentage  of  substances  usually  regarded  as  essentially  of  nuclear 
origin.  It  is  quite  certain,  therefore,  that,  although  there  may  be  no 
morphologic  nucleus  demonstrable  by  ordinary  staining  methods, 
nuclear  material  is  present  in  abundance  in  the  organism. 

Metachromatic  Granules. — Certain  types  of  bacteria,  notably  mem- 
bers of  the  diphtheria  and  hemorrhagic  septicemia  groups,  exhibit 
one  or  more  highly  refractile  granules  in  an  otherwise  homogeneous 
endoplasm  when  they  are  examined  unstained  with  the  higher  powers 
of  the  microscope.  These  granules  are  few  in  number  in  the  diphtheria 
bacillus  group  and  are  distributed  somewhat  irregularly  throughout 
the  cell,  one  or  more  granules  usually  being  greater  in  diameter  than  the 
cell  itself,  thus  giving  the  rod  a  swollen  appearance.  In  the  hemor- 
rhagic septicemia  group  these  granules  are  arranged  symmetrically, 
one  at  each  end  of  the  organism,  polar  granules.  Such  granules  are 
called  Ernst-Babes  or  metachromatic  granules.  They  color  differently 
from  the  rest  of  the  cell  when  they  are  stained  with  methylene  blue, 
appearing  as  mahogany-red  spots  in  the  deep  blue  endoplasm.  They 
retain  the  stain  rather  tenaciously.  Many  theories  have  been  advanced 
to  explain  their  significance,  but  nothing  definite  is  known  about  them, 
except  that  these  granules  appear  to  differ  widely  in  chemical  composi- 
tion. Some  are  colored  brown  with  iodine,  suggesting  that  they  may 
be  related  to  glycogen.3  Some  stain  black  with  osmic  acid,  suggesting 

1  Centralbl.  f.  Bakteriol.,  1901,  xxx,  97,  145,  193,  225. 

2  Ztschr.  f.  Hyg.,  1899,  xxx,  1;   Festschr.  z.  60  Geburtstage  vonR.  Koch,  1903,  p.  383. 

3  A.  Meyer,  Flora,  1899,  Ixxxvi,  428. 


28  THE  MORPHOLOGY  OF  BACTERIA 

that  they  may  be  fatty  or  lipoidal  in  composition,  while  others  are 
probably  complex  phosphorus-containing  compounds.1  Not  all  of  these 
varieties  of  granules  are  met  with  in  the  same  organism.2  Among 
the  higher  bacteria  granules  of  sulphur  or  of  iron  are  demonstrable 
respectively  in  the  sulphur  and  the  iron  bacteria. 

Flagella. — All  minute  particles  suspended  in  water  or  other  fluids 
of  low  viscosity  are  in  constant  motion.  This  motion,  which  is  irregular 
and  tremulous,  was  first  described  by  Brown:3  it  is  variously  termed 
Brownian  movement,  pedesis,  or  molecular  movement.  Brownian 
movement  may  be  rapid  or  slow,  extensive  or  circumscribed,  depending 
upon  the  nature  of  the  particles  and  the  composition  and  temperature 
of  the  fluid  in  which  they  are  suspended.  This  is  not  true  motility, 
even  though  each  individual  particle  moves  independently  of  the  other 
particles  in  an  irregular  orbit,  for  the  particles  as  a  whole  do  not 
permanently  change  their  relative  positions.  Dead  bacteria  and  many 


FIG.  2. — Flagella.     1'and  6,  peritrichic  flagella;   2  and  4,  monotrichic  flagella;   3  and  5, 

lophotrichic  flagella. 

living  bacteria,  notably  the  cocci,  exhibit  the  Brownian  movement. 
Many  bacilli  and  spirilla,  on  the  contrary,  possess  the  power  of 
independent  motility,  that  is,  they  can  progressively  and  permanently 
change  their  relative  positions  in  space.  Motile  bacteria  are  provided 
with  one  or  more  long,  delicate,  contractile  filaments — flagella — which 
are  probably  the  organs  of  locomotion.  These  flagella  cannot  be 
demonstrated  on  living  bacteria,  except  possibly  by  dark-ground  illu- 
mination, and  ordinary  staining  reactions  usually  fail  to  reveal  them. 
Special  staining  methods  show  them  clearly.  They  appear  to  arise 
from  the  cell  membrane.4  Their  arrangement  and  number  is  varied 
among  bacteria  in  general,  but  relatively  constant  for  a  particular 
variety  of  bacterium:  they  are  thinner  as  a  rule  on  younger  bacterial 
cells,  thicker  on  older  organisms.5  A  cholera  vibrio  has  a  single 

1  Grimme,  Centralbl.  f.  Bakteriol.,  1904,  xxxvi,  952. 

2  For  literature  see  Marx  and  Woithe,  Centralbl.  f.  Bakteriol.,  1900,  xxviii,  1,  33,  65, 
97;   Krompecher,  ibid.,  1901,  xxx,  385,  425;   Gauss,  ibid.,  1902,  xxxi,  92. 

3  Edinburgh  Phil.  Jour.,  1828,  v,  358;  1830,  viii,  41. 

4  Schaudinn,  Arch.  f.  Protistenk.,  1903,  i,  421. 

6  De  Grandi,  Centralbl.  f.  Bakteriol.,  1903,  xxxiv,  97. 


STRUCTURE  AND  CONSTITUENTS  OF  BACTERIAL  CELL       29 

flagellum  at  one  or  both  ends  of  the  organism;  in  the  typhoid  bacillus 
they  are  distributed  around  the  sides  of  the  organism  but  do  not  occur 
at  the  ends. 

Spores. — Endospores. — Many  bacteria  die  when  their  environment 
becomes  unsuited  for  further  growth.  Death  may  result  from  the 
presence  of  inimical  substances,  the  absence  of  essential  foods,  or  the 
intervention  of  unsuitable  physical  conditions.  Death  is  manifested 
by  a  cessation  of  chemical  interchange  between  the  bacterial  cell  and 
its  environment.  There  is  a  group  of  bacteria,  however,  usually  of 
saprophytic  origin,  which  is  able  to  survive  even  prolonged  exposure 
to  unfavorable  environmental  conditions  by  passing  into  a  latent  stage 
during  which  chemical  interchange  with  the  environment  is  at  an 
extremely  low  ebb.  This  latent  stage  or  hibernation  has  been  known 
to  last  for  more  than  two  decades  in  certain  instances,  and  yet  the 
organisms  have  resumed  their  original  luxuriant  growth  when  placed 


<*> 


FIG.  3.  —  Types  of  bacterial  spores. 

under  favorable  conditions.  The  bacteria  which  exhibit  this  latent 
state  produce  within  their  substance  highly  refractile,  spherical  or  oval 
bodies  called  spores.  Spores  are  not  found  in  very  young,  actively 
growing  cultures,  as  a  rule.  Spore  formation  is  ushered  in  by  a  clouding 
of  the  endoplasm  of  the  bacterial  cell,  which  gradually  becomes  granular. 
The  granules  coalesce,  eventually  appearing  as  the  mature  spore  which 
is  surrounded  by  a  dense  membrane,  frequently  exhibiting  a  double 
contour  when  stained  by  dilute  carbol  fuchsin.1  The  spore  membrane 
(ectoplasm)  is  relatively  impermeable  to  heat  and  disinfectants  and 
confers  the  resistance  to  physical  agents  which  spores  exhibit  upon 
them.  But  one  spore  is  formed  in  an  individual  bacterium,  except 
under  most  unusual  conditions.  It  is  to  be  emphasized,  consequently, 
that  spore  formation  is  not  a  reproductive  process.  The  mature  spore 
may  form  in  the  center  of  the  bacterium,  at,  or  near  one  end.  The 
spore  may  be  round  or  oval,  and  greater  or  lesser  in  diameter  than 
the  parent  cell.  If  the  spore  is  greater  in  diameter  it  distends  the 
cell  membrane,  producing  a  spindle-shaped  organism  if  the  spore  is 

1  Meyer,  A.,  Practicum  der  botanischen  Bakterienkunde,  Jena,  1903. 


30  THE  MORPHOLOGY  OF  BACTERIA 

in  the  center  of  the  rod :  if  the  spore  is  at  one  end,  a  drumstick-shaped 
organism  results.  Usually  the  size  and  position  of  the  spore  is  fairly 
constant  in  a  given  type  of  bacteria.  Spore  formation  is  most  common 
among  the  anaerobes,  fairly  common  among  the  saprophytic  bacteria, 
practically  absent  in  the  pathogenic  bacteria,  and  practically  never 
takes  place  spontaneously  in  the  human  or  animal  body.  The  spiral 
organisms  rarely  produce  spores,  and,  with  the  exception  of  Sarcina 
pulmonum,  spore  formation  is  practically  never  observed  in  the  cocci. 

It  has  never  been  satisfactorily  determined  whether  spore  formation 
is  a  regular  definite  stage  in  the  life  history  of  bacteria  which  produce 
them  or  whether  spores  are  produced  rather  under  the  stress  of  unfavor- 
able evironmental  conditions. 

Germination  of  Spores. — When  bacterial  spores  are  placed  in  an 
environment  favorable  to  the  vegetative  activity  of  the  cell,  they 
germinate:  the  dense  membrane  which  constitutes  the  ectoplasm  of 
the  spore  softens,  usually  at  the  pole  or  the  equator,  and  the  vegeta- 
tive rod  emerges,  at  first  as  a  small  bud,  then  rapidly  assumes  the 
typical  size  and  shape  of  the  fully  mature  cell.  The  development  of 
the  anthrax  bacillus  from  the  spore  is  usually  in  the  line  of  the  longer 
axis,  polar  germination:  B.  subtilis,  on  the  contrary,  usually  emerges 
at  right  angles  to  the  larger  axis  of  the  spore,  equatorial  germination. 
Many  spores  are  circular  in  outline,  and  in  such  cases  the  relation  of  the 
developing  vegetative  cell  to  the  axis  of  the  spore  is  unknown.  Fre- 
quently the  remnants  of  the  spore  membrane  remain  attached  to  one 
end  of  the  newly  formed  vegetative  cell,  appearing  as  a  cap,  as  it  were. 
Some  spores  do  not  appear  to  rupture  as  germination  takes  place.— 
the  newly  forming  organism  appears  to  absorb  the  entire  spore  and 
its  ectoplasm,  incorporating  the  entire  structure  by  solution  in  the 
vegetative  cell. 

Arthrospores. — Certain  organisms  belonging  to  the  coccal  group, 
more  particularly  the  streptococci,  exhibit  from  time  to  time  cells 
which  are  decidedly  larger  than  their  fellows.  These  cells  are  more 
highly  refractile,  they  usually  possess  a  granular  cytoplasm,  and  fre- 
quently stain  somewhat  irregularly.  They  have  been  designated  by 
Hueppe  as  arthrospores.  These  arthrospores  appear  to  have  no 
unusual  resisting  powers,  and  they  are  in  no  sense  to  be  regarded  as 
true  spores.  It  is  very  probable  that  they  are  involution  forms. 


REPRODUCTION  AND  CELL  DIVISION  31 

E.     REPRODUCTION   AND   CELL  DIVISION. 

Bacteria  are  structurally  the  simplest  known  organisms  which  main- 
tain an  independent  existence:  all  their  vital  functions  are  exhibited 
in  a  single  asexual  cell  devoid  of  a  morphologically  definable  nucleus. 
The  absence  of  sexual  characters  and  of  a  morphologic  nucleus  makes 
bacterial  reproduction  mechanically  a  simple  process,  and  doubtless 
the  rapid  sequence  of  generations  observed  in  various  bacteria  depends 
in  part  upon  this  simplicity  of  structure. 

Reproduction  takes  place  in  the  following  manner:  A  bacterial 
cell  placed  in  a  favorable  environment  increases  in  size  until  it  reaches 
a  maximum  which  is  relatively  constant  for  each  variety;  then  a  slight 
equatorial  constriction  occurs,  which  deepens  until  a  distinct  septum 
is  produced  by  invagination,  which  divides  the  original  cell  into  two 
morphologically  complete,  fully  mature  individuals  of  approximately 
equal  size.  It  is  obvious  that  this  septum  consists  ordinarily  of  at 
least  two  layers,  since  one  layer  is  required  to  complete  each  of  the 
dividing  individuals.  Successive  generations  may  be  produced  at 
intervals  which  may  be  as  frequent  as  every  fifteen  minutes  in  the 
more  rapidly  growing  types.  Septation  usually  takes  place  deliberately  ; 
that  is  to  say,  the  septum  forms  relatively  slowly.  Diptheria  bacilli 
and  possibly  related  bacteria  divide  somewhat  differently;  the  parental 
cell  appears  to  be  under  tension  when  the  septum  becomes  visible, 
and  the  daughter  cells  spring  apart  suddenly  when  septation  is  com- 
pleted. So  forcible  is  this  separation  that  the  daughter  cells  lie  at  an 
angle  with  each  other:  Nakanishi1  has  observed  that  the  septum  in 
this  group  of  organisms  frequently  forms  at  a  metachromatic  granule. 
Septation  in  the  Bacillacese  and  Spirillacese  normally  takes  place 
at  right  angles  to  the  long  axis  of  the  organism,  and  midway  between 
the  ends,  thus  effecting  the  separation  into  two  individuals  with  the 
minimal  expenditure  of  material;  in  the  Coccacese,  which  are  usually 
isodiametric,  no  economy  of  material  in  septation  is  apparent,  and  no 
known  force  determines  the  initial  plane  of  septation:  subsequent 
fission  may  be  definitely  related  to  the  initial  plane.  Noguchi2  has 
brought  forward  striking  evidence  and  photographic  illustrations  in 
favor  of  the  view  that  the  Spirocheta  (Treponemata)  may  reproduce 
by  longitudinal  fission  rather  than  by  transverse  fission.  If  this  view 
be  generally  adopted,  it  would  contradict  the  "minimal  requirement 

1  Centralbl.  f.  Bakteriol.,  1900,  xxvii,  641. 

2  Jour.  Exper.  Med.,  1912,  xv,  201. 


32  THE  MORPHOLOGY  OF  BACTERIA 

theory,"  which  assumes  that  transverse  fission,  the  more  economical 

process  both  with  respect  to  amount  of  material  and  expenditure 

of   energy,  holds  universally  for   bacteria,  as  has  previously  been 
maintained. 

F.     CELL   GROUPING. 

In  Bacilli  and  Spirilla,  where  septation  typically  occurs  at  right 
angles  to  the  long  axis  of  the  organism,  it  is  obvious  that  no  geometrical 
arrangement  of  cells  is  possible  other  than  the  formation  of  chains  of 
rods  or  of  spirals  if  the  individual  organisms  remain  adherent.  The 
cocci,  on  the  other  hand,  are  spherical  and  have  no  longer  or  shorter 
axis,  consequently  a  definite  sequence  of  septation  in  one,  two  or 
three  planes  of  space  can  give  rise  to  (1)  chains  of  cocci,  if  the  plane 
of  septation  is  always  in  one  plane  of  space;  (2)  groups  of  four  cocci, 
if  septation  takes  place  alternately  in  two  planes  of  space;  or  (3)  in 
packets  of  cocci,  if  septation  is  alternate  in  three  planes  of  space. 
Many  cocci  do  not  exhibit  a  definite  sequence  of  planes  of  septation. 

G.     CLASSIFICATION   OF   BACTERIA. 

Relation  to  Higher  Plants.— The  position  of  Bacteria  in  the  Plant 
Kingdom  is  indicated  in  the  following  table : 

Plant  Kingdom. 


Cryptogamia 
Thallophyta 

Phanerogamia 

Algae 

Schizomycetes 
(Bacteriacese) 

1 

Fungi 

Saccharomycetes 
Blastomycetes 

(yeasts) 

i 
Lichens 
1 
Hyphomycetes 
(molds) 

Eubacteriaceae 
Coccacese 
Bacillacese 
Spirillacese 

Chlamydobacteriaceae 
Streptothrix 
Phragmidothrix 
Crenothrix 
Cladothrix 
Actinomycetes 

A  complete  natural  classification  of  bacteria  is  impossible  at  the 
present  time.  The  monotony  of  form  observed  in  this  group  of 
organisms  merely  suffices  to  classify  them  into  three  great  divisions: 
Cocci,  Bacilli,  and  Spirilla.  Further  subdivision  into  groups  which 
are  potentially  families,  genera  and  species  is  accomplished  by  arrang- 


CLASSIFICATION  OF  BACTERIA  33 

ing  them  according  to  their  physiological  and  chemical  activities. 
Even  this  artificial  procedure  is  unsatisfactory,  for  bacteriological 
diagnosis  is  a  subject  which  has  developed  under  the  stress  of  practical 
needs,  and  as  bacteria  play  a  part  in  many  fields  of  activity,  it  has 
inevitably  followed  that  the  criteria  whereby  they  are  recognized 
vary  greatly  according  to  the  art  or  science  in  which  they  are  contem- 
plated. Even  the  same  species  may  be  identified  by  wholly  different 
characteristics.  Notwithstanding  the  difficulties  which  surround  the 
grouping  of  bacteria,  Migula1  has  worked  out  a  system  of  classification 
based  upon  purely  morphological  characteristics,  which  effects  a 
primary  separation  of  bacteria  into  smaller  subdivisions,  which  is 
moderately  satisfactory  so  far  as  it  goes,  and  it  is  the  one  commonly 
adopted. 
With  certain  additions  it  is  as  follows: 

THE  TRUE  BACTERIA:  EUBACTERIACE.E. 

1.  Coccacece.     Cells  in  the  free  state  spherical. 

(a)  Micrococcus.     Cells  spherical.    No  definite  sequence  of  planes  of  septa- 

tion. 
(6)  Diplococcus.    Organisms  habitually  occur  in  pairs. 

(c)  Streptococcus.     Plane  of  septation  parallel.     Form  longer  or  shorter 

chains. 

(d)  Tetracoccus.    Planes  of  septation  alternate,  and  at  right  angles  in  two 

planes  of  space.    Form  groups  of  four  or  tetrads. 

(e)  Sarcina.    Planes  of  septation  alternate,  at  right  angles,  in  three  planes 

of  space.      Form  packets. 

(/)   Planococcus.    Motile  cocci,  provided  with  flagella. 
(g)  Planosarcina.     Motile  sarcina,  provided  with  flagella. 

2.  Bacillaceoe.     Cells  elongated  and  cylindrical;  straight. 

(a)  Bacterium.     Non-motile.     No  flagella. 
(6)  Bacillus.    Cells  motile.    Peritrichic  flagellation. 

(c)   Pseudomonas.     Cells  motile.     Polar  flagellation.     Single  flagellum  or 
tufts  of  flagella  at  one  or  both  poles  of  the  organism. 

3.  Spirillacece.    Cells  elongated  and  cylindrical;  spirally  twisted  about  the  long 

axis. 

(a)  Spirasoma.    Cells  rigid  and  slightly  curved;  without  flagella. 
(6)  Microspira.     Cells  rigid  and  slightly  curved;  with  one,  rarely  several, 

polar  flagella. 

(c)  Spirillum.    Cells  rigid,  loosely  coiled;  with  tuft  of  polar  flagella. 

(d)  Spirocheta.     Cells  flexuous,  closely  coiled;  flagellation  unknown. 

THE  HIGHER  BACTERIA. 

4.  Chlamydobacteriacece.    Cells  enclosed  in  a  sheath. 

(a)  Streptothrix.    Cell  division  always  in  one  plane. 

(6)  Phragmidothrix.    Cell  division  in  three  planes  of  space;  very  delicate 
sheath. 

(c)  Crenothrix.  Cell  division  in  three  planes  of  space;  sheath  well  developed. 

(d)  Cladothrix.     Cells  more  or  less  branched. 

5.  Beggiatoacece  (Thiothrix).    Cells  contain  sulphur  granules. 

1  System  d.  Bakterien,  Jena,  1907. 


34  THE  MORPHOLOGY  OF  BACTERIA 

H.     MUTATION:   CONSTANCY   OF   TYPES.1 

True  mutation  or  discontinuous  variation  is  rarely  observed  among 
bacteria,  although  a  few  instances  are  on  record  which  have  been  sub- 
jected to  satisfactory  scrutiny.  Mutation  must  be  carefully  differen- 
tiated from  the  loss  of  one  or  more  characteristics  of  bacteria  during 
cultivation;  the  loss  or  suppression  of  one  or  more  characteristics  is 
fairly  commonly  observed  among  bacteria.  Pigment  production,  and 
proteolytic  activity — as  for  example  the  ability  to  liquefy  gelatin- 
are  frequently  lost  to  cultures  of  bacteria  during  prolonged  cultiva- 
tion, but  these  properties  may  be  regained  when  the  organisms  are 
placed  once  more  in  a  suitable  environment.  Similarly,  strains  of 
fermenting  bacteria  may  temporarily,  or  even  permanently,  become 
unable  to  decompose  certain  carbohydrates.  Change  in  virulence, 
or  loss  of  virulence  is  rather  commonly  noticed  among  pathogenic 
bacteria  grown  outside  the  animal  body.  It  is  even  possible  to  so 
parasitize  organisms  by  prolonged  cultivation  upon  one  medium  that 
they  will  develop  not  at  all,  or  slowly  at  best,  on  other  media.  Thus, 
a  strain  of  B.  proteus  has  been  grown  continuously  upon  agar  with 
frequent  transfers  for  four  years,  and  the  organism  will  no  longer 
grow  in  broth.  Similarly,  B.  bulgaricus  is  an  obligate  milk  parasite. 
Exposure  to  unfavorable  environmental  conditions  may  also  suppress 
important  characters:  Pasteur's  celebrated  experiment  of  growing 
anthrax  bacilli  at  43°  C.  for  some  hours  and  establishing  an  asporeless 
variety  is  a  familiar  example.  The  suppression  of  characters  as  out- 
lined above  is  frequently  important  as  the  starting  point  for  new 
adjustments  between  pathogenic  bacteria  and  their  hosts. 

Turning  to  the  production  of  disease  in  man,  it  is  certain  that  at 
least  some  organisms  produce  the  same  reaction  today  they  did  years 
ago :  tuberculosis  appears  to  be  the  same  disease  today  it  was  centuries 
ago,  as  is  evidenced  by  the  lesions  found  in  Egyptian  mummies.  Clini- 
cally, the  observations  of  Hippocrates  would  be  a  fair  exposition  of  the 
phenomena  seen  in  tuberculous  patients  at  the  present  time.  Leprosy 
also  appears  to  be  the  same  entity  now  it  was  during  the  middle  ages, 
although  the  geographical  distribution  is  much  more  restricted.  With 
respect  to  more  acute  diseases,  which  require  more  careful  examination 
to  differentiate  them,  the  evidence  is  less  certain,  although  typhoid 
bacilli  do  not  appear  to  have  changed  since  they  were  first  isolated 

1  Eisenberg,  Ueber  Mutationen  bei  Bakterien  und  anderen  Mikroorganismen  in 
Ergebnisse  d.  Immunitatsforsch.  experimentellen  Therapie,  Bakteriologie  und  Hygiene, 
Berlin,  1914,  pp.  28-142,  for  summary. 


MUTATION:  CONSTANCY  OF  TYPES  35 

by  Gaffky  three  decades  ago.  It  appears  to  be  reasonably  certain 
from  what  is  known  of  bacteria  and  the  manifestations  of  disease 
they  induce  that  mutation  is  an  infrequent  phenomenon:  attenuation 
and  the  partial  suppresion  of  characteristics,  on  the  contrary,  appear 
to  be  quite  common.  The  available  evidence  indicates  that  bacterial 
types  are  stabile  under  natural  conditions :  there  is  no  definite  evidence 
in  favor  of  the  view  that  bacteria  change  slowly  or  abruptly  either  in 
their  morphology  or  in  the  changes  they  induce  in  their  environment 
in  the  sense  that  entirely  new,  unrelated  types  are  developed  de  now 
from  preexisting  types.  This  does  not  preclude  the  possibility  that 
such  changes  have  taken  place  in  the  past,  rather  that  such  changes, 
if  they  have  taken  place,  have  not  been  definitely  established. 


CHAPTER  II. 


GENERAL  PHYSIOLOGY  OF  BACTERIA— THE  EFFECT 
OF  ENVIRONMENT  ON  BACTERIA. 


A.  RATE  OF  REPRODUCTION. 

B.  MOTILITY:  RATE  OF  MOTION. 

C.  SPORULATION:    GERMINATION    OF 

SPORES. 

D.  LONGEVITY. 

E.  MOISTURE:  DESICCATION. 

F.  OXYGEN.     AEROBIOSIS  AND  ANAERO- 

BIOSIS. 

G.  TEMPERATURE. 

1.  General. 

2.  Cold. 

3.  Heat. 

H.  HEAT  PRODUCTION. 

I.    LIGHT  AND  ELECTRICITY. 


J.    GRAVITY,  OSMOTIC  PRESSURE,  AGI- 
TATION AND  CHEMOTAXIS. 
K.  ENZYMES,   TOXINS.     PTOMAINS. 
L.   PIGMENTS. 

1.  Photodynamic. 

2.  Phosphorescent. 

3.  Fluorescent. 

4.  Chromogenic. 

M.  SYMBIOSIS,  ANTIBIOSIS,  COMMENSAL- 
ISM. 

N.  MEDIA — COMPOSITION  AND  REAC- 
TION. 

O.  GROWTH  IN  ANIMAL  BODY. 


A.     RATE  OF  REPRODUCTION. 

ONE  of  the  striking  characteristics  of  the  Bacteriacese  is  their  rapidity 
of  reproduction.  Among  the  most  actively  growing  types  of  bacteria, 
as,  for  example,  the  cholera  vibrio,  successive  generations  may  appear 
at  intervals  as  frequent  as  every  fifteen  minutes  when  the  environ- 
mental conditions  are  most  favorable :  that  is  to  say,  ninety-six  genera- 
tions are  theoretically  possible  in  twenty-four  hours.  If  this  rate  of 
reproduction  could  be  maintained  for  three  days,  the  progeny  of  a 
single  organism  would  occupy  a  space  not  less  than  that  of  the  com- 
bined waters  of  the  earth.  Fortunately,  nature  imposes  many  restraints 
which  limit  the  numbers  of  bacteria.  The  rapid  accumulation  of  waste 
products,  the  exhaustion  of  nutrient  material,  and  the  enormous 
death  rate  in  culture  media  even  after  a  comparatively  few  hours' 
growth,  together  with  other  factors  restrict  development  to  such  a 
degree  that  the  actual  number  of  living  descendants  of  bacteria  in 
cultures  or  in  nature  falls  far  short  of  the  theoretical  number.  Many 
bacteria  develop  more  slowy  than  this,  however.  They  may  require 
hours  or  even  days  to  arrive  at  maturity.  The  tubercle  bacillus,  for 
example,  grows  comparatively  slowy  in  artificial  media  (where  such 
observations  are  of  necessity  made),  and  the  frequency  of  septation, 
even  in  the  most  rapidly  growing  bacteria,  is  greatly  affected  by 
environmental  factors. 


SPORULATION:   GERMINATION  OF  SPORES  37 

Generally  speaking,  when  nutritional  conditions  are  favorable,  the 
rate  of  reproduction  is  influenced  by  temperature,  growth  being  most 
rapid  when  the  temperature  is  optimum  for  the  organism,  less  rapid 
when  the  temperature  exceeds  or  falls  below  this  point. 

B.     MOTILITY:    RATE  OF  MOTION. 

The  rhythmic  contractions  of  the  flagella,  with  which  practically 
all  motile  bacteria  are  provided,  drive  the  organisms  through  fluid 
media  in  which  they  may  be  suspended,  some  slowly,  some  rapidly. 
Not  all  bacteria  even  in  the  same  culture  exhibit  motility.  The  char- 
acter of  the  motion  may  be  direct,  serpentine,  oscillatory,  or  irregular. 
Rarely,  the  flagella  appear  to  produce  local  currents  in  the  medium 
which  immediately  surrounds  the  organism.  Various  environmental 
factors  incite  or  inhibit  motility.  Chemotactic  substances  may  attract 
bacteria,  thus  in  a  sense  directing  their  line  of  movement.  Other 
substances,  as  protoplasmic  poisons,  paralyze  bacterial  movements. 
Oxygen  appears  to  increase  the  motility  of  aerobic  bacteria,  and  it 
inhibits  motility  in  the  anaerobes.  Generally  speaking,  in  favorable 
media  motility  increases  with  the  rise  in  temperature  to  the  optimum. 
If  this  temperature  is  exceeded,  even  by  a  very  few  degrees,  motion 
ceases. 

The  rate  at  which  bacteria  progress  through  a  fluid  is  a  variable 
one,  although  with  a  given  organism  under  favorable  conditions  it 
appears  to  be  fairly  constant.  It  must  be  remembered  that  the 
apparent  rate  of  motion  observed  under  the  microscope  is  increased 
proportionately  to  the  increase  in  magnification.  Lehmann  and 
Fried1  have  measured  the  average  speed  of  certain  bacteria  in  fluid 
media  in  millimeters  per  second.  They  find  that  of  the -cholera  vibrio 
to  be  0.03,  typhoid  bacillus  0.018,  B.  subtilis  0.01,  B.  megatherium 
0.0075.  If  a  man  traveled  at  a  rate  of  speed  in  proportion  to  his  size 
as  great  as  that  of  the  cholera  vibrio,  he  would  average  more  than  a 
mile  a  minute. 

C.     SPORULATION:    GERMINATION  OF  SPORES. 

Many  saprophytic  bacteria  form  within  themselves  spores  which 
appear  apparently  under  the  stimulus  of  the  stress  of  conditions  unfa- 
vorable for  the  continued  vegetative  growth  of  the  organism.  Sporula- 
tion,  in  other  words,  appears  to  be  a  specialized  mechanism  for  the 

1  Arch.  f.  Hyg.,  1903,  xlvi,  314. 


38  GENERAL  PHYSIOLOGY  OF  BACTERIA 

perpetuation  of  the  organism  during  periods  of  environmental  unfit- 
ness.  Whether  spore  formation  is  a  definite  phase  in  the  life-history  of 
spore-forming  bacteria  is  not  definitely  settled.  Sporulation  is  rarely 
observed  when  the  temperature  of  the  environment  falls  much  below 
15°  C.,  although  considerable  latitude  is  observed  among  the  spore- 
forming  bacteria  in  this  respect.  Spores  are  rarely,  if  ever,  produced 
within  the  tissues  of  the  animal  body:  if  the  tissues  are  exposed  to 
the  air,  however,  particularly  postmortem,  spore  formation  may  take 
place.  No  bacteria  progressively  pathogenic  for  man  are  known  to 
form  spores. 

The  unusual  resistance  of  mature  spores  to  desiccation,  to  exposure 
to  dry  and  moist  heat,  and  to  disinfectants  may  be  due  either  to 
their  low  content  of  water,  for  spores  contain  less  than  half  of  the 
water  contained  in  the  normal  vegetative  cell,  to  the  relatively  thick 


o 


FIG.  4. — Germination  of  bacterial  spores.     1,  by  absorption  of  spore  membrane; 
2,  equatorial  germination;   3,  polar  germination. 

refractile  spore  membrane,  or  to  unusual  concentrations  of  fatty  and 
lipoidal  substances.  Experiments  by  Lewith1  would  suggest  that  the 
relative  desiccation  of  the  contents  of  spores  as  compared  with  the  mois- 
ture content  of  the  vegetative  organism  would  be  the  most  plausible 
explanation  of  their  resistance  to  heat  without  apparent  injury.  He 
found  that  egg  albumen  (dried)  suspended  in  5  per  cent,  of  water  coagu- 
lated at  145°  C.;  suspended  in  18  per  cent,  of  water,  coagulation  took 
place  at  90°  C.;  with  25  per  cent,  of  water,  at  80°  C.;  and  in  a  consider- 
able volume  of  water  (amount  not  stated)  coagulation  occurred  when 
the  temperature  reached  56°  C. 

The  resistance  of  spores  to  physical  conditions  varies  somewhat 
according  to  the  organism  in  which  they  are  formed.  Generally 
speaking,  however,  several  minutes'  exposure  to  the  temperature 
of  boiling  water  (100°  C.)  may  fail  to  kill  them.  Dry  heat  is  less 
effective  than  moist  heat,  for  an  exposure  of  160°  C.  for  one  and  one- 
half  hours  is  required  to  certainly  sterilize  glassware  containing  spores. 
Ten  to  fifteen  pounds  live  steam  pressure  for  fifteen  minutes  is  required 

1  Arch.  f.  exp.  Path.  u.  Pharmakol.,  1890,  xxvi. 


MOISTURE  AND  DESICCATION  39 

to  effect  sterilization  of  liquids  and  organic  matter  in  general.    Direct 
sunlight  will  kill  spores  after  days  of  exposure. 

Germination  of  bacterial  spores  takes  place  when  they  are  placed 
in  a  suitable  nutritive  environment  in  which  the  temperature,  moisture 
and  oxygen  relations  are  favorable.  The  vegetative  cell  breaks  through 
the  spore  membrane  apparently  after  the  latter  has  lost  its  refractility, 
and  reproduction  by  fission  proceeds  anew,  and  persists  until  environ- 
mental conditions  again  lead  to  sporulation. 

D.     LONGEVITY. 

The  duration  of  life  in  the  individual  non-spore-forming  bacterium 
is  unknown,  but  it  is  greatest  apparently  when  the  organism  is  quiescent 
or  nearly  so.  This  condition  is  realized  most  commonly  when  bacteria 
are  exposed  to  temperatures  slightly  above  freezing  in  a  dark  place. 
This  question  has  been  studied  recently  under  unusual  conditions. 
A  mastodon  was  discovered  in  Siberia  which  had  been  uncovered  by 
an  unusual  recession  of  the  ice.  This  animal  was  found  to  be  practi- 
cally intact,  and  cultures  made  with  proper  precautions  from  the 
center  of  the  proboscis  contained  bacteria  indistinguishable  from 
Sarcina  lutea  and  other  well  known  air  organisms.1  If  these  cultures 
are  authentic,  a  most  unexpected  instance  of  bacterial  longevity  has 
been  unearthed,  for  this  animal  has  undoubtedly  been  frozen  for 
hundreds  of  years. 

Spores  have  been  dried  and  kept  in  a  cool  dry  place  for  more  than 
two  decades,  and  yet  developed  with  their  usual  luxuriance  when  placed 
in  a  favorable  environment.  Dried  anthrax  spores  thus  retain  not  only 
their  viability  but  their  virulence  unimpaired  for  years.  Practically, 
the.  average  duration  of  life  among  bacteria  is  comparatively  brief. 

E.     MOISTURE  AND  DESICCATION. 

Bacteria  normally  contain  at  least  80  per  cent,  of  moisture  in  their 
substance,  and  they  develop  typically  only  in  media  containing  con- 
siderable amounts  of  moisture.  Bacteria  do  not  vegetate  normally 
in  desiccated  media,  but  many  varieties  resist  drying  for  considerable 
periods.  Advantage  is  taken  of  the  restriction  of  bacterial  develop- 
ment in  the  absence  of  suitable  amounts  of  moisture  in  various  pro- 
cesses of  drying  meats  and  other  foodstuffs;  desiccated  foods  will 
keep  for  weeks  under  the  proper  conditions.  Bacterial  spores  pro- 

1  Russian  Academy  of  Science,  1911-1912. 


40  GENERAL  PHYSIOLOGY  OF  BACTERIA 

tected  from  direct  sunlight  are  extremely  resistant  to  drying,  but 
they  develop  with  characteristic  vigor  when  environmental  conditions 
become  suitable.  Even  non-sporogenic  bacteria  may  develop  after 
days  or  weeks  of  desiccation.  Many  pathogenic  bacteria  are  eliminated 
from  the  body  enveloped  in  albuminous  material,  as  in  sputum.  These 
organisms  thus  protected  may  resist  drying  for  many  days,  provided 
they  are  not  exposed  to  direct  light.  The  following  table  indicates 
the  relative  viability  of  various  bacteria  pathogenic  for  man  to  air 
drying.1 

1.  Gonococcus,  few  hours. 

2.  Cholera  vibrio,  few  hours  to  two  days. 

3.  Plague  bacillus,  one  to  eight  days. 

4.  Diphtheria  bacillus,  twenty  to  thirty  days. 

5.  Streptococcus  pyogenes,  fourteen  to  thirty-six  days. 

6.  Pneumococcus,  nineteen  to  fifty-five  days. 

7.  Staphylococcus  pyogenes,  fifty-five  to  one  hundred  days. 

8.  Typhoid  bacillus,  up  to  seventy  days. 

9.  Tubercle  bacillus,  two  to  three  months. 

F.    OXYGEN:  AEROBIOSIS  AND  ANAEROBIOSIS. 

Oxygen,  either  in  the  free  state  or  combined,  is  essential  to  the 
growth  of  all  known  bacteria.  The  majority  of  bacteria  grow  best  in 
the  presence  of  free  (atmospheric)  oxygen,  although  the  percentage 
of  this  gas  necessary  to  support  bacterial  life  may  be  considerably 
less  than  that  occurring  normally  in  the  air.  Some  bacteria  appear 
to  be  wholly  dependent  upon  free  oxygen,  and  they  are  called  obligate 
aerobes.  A  small  group  of  bacteria,  on  the  contrary,  grow  only  in  the 
absence  of  free  oxygen,  and  more  than  minimal  concentrations  of  this 
gas  are  actually  poisonous  to  them.  Those  bacteria  which  grow  only 
in  the  absence  of  free  oxygen  are  called  obligate  anaerobes.  The  vast 
majority  of  bacteria  are  facultative  with  respect  to  their  oxygen 
requirement,  growing  best  in  the  presence  of  atmospheric  oxygen  but 
able  to  develop  either  in  the  presence  of  small  amounts  of  free  oxygen, 
as  in.  the  tissue  of  the  body  and  certain  parts  of  the  intestinal  tract, 
or  they  are  able  to  obtain  their  oxygen  from  chemical  compounds,  as 
certain  simple  sugars,  if  free  oxygen  is  not  available.  These  organisms 
are  called  facultative  anaerobes.  The  maximum  tolerance  of  bacteria 
for  oxygen  varies  very  considerably,  as  the  following  table  indicates: 

Oxygen  content  of  the  air  is  taken  as  100  per  cent. 

1  Fischer,  Vorlesungen  iiber  Bakterien  1903,  II  Aufl.,  110. 


TEMPERATURE  41 

MAXIMUM    OXYGEN   TOLERANCE. 

Atmospheric  oxygen, 
Per  cent. 

B.  (clostridium)  butyricus     ...........  1  .  35 

B.  chauvei  ................  5.00 

B.  edematis  maligni    ...........      .      .  3.25 

Purple  bacteria  (Molisch)       .........  about  90.00 

Thiosulphate  bacteria  (Nathansson)      ........  400.00 

B.  prodigiosus        ..............  3000.00 

G.    TEMPERATURE. 

1.  General.  —  The  extreme  temperature  limits  of  bacterial  growth 
are  very  slightly  above  0°  C.  to  80°  C.  inclusive.  Some  bacteria, 
notably  those  found  in  the  Arctic  regions,  appear  to  develop  even  at 
0°  C.;  others,  chiefly  those  found  in  soil,  feces,  and  certain  thermal 
springs,  grow  even  at  80°  C.,  a  degree  of  heat  considerably  above  that 
at  which  the  protoplasm  of  most  animals  and  plants  is  coagulated. 
The  vast  majority  of  bacteria,  however,  develop  best  within  a  range 
of  temperature  from  15°  C.  as  a  minimum  to  40-43°  C.  as  a  maximum. 
All  bacteria  exhibit  three  cardinal  thermic  points  :  a  minimum  tempera- 
ture, below  which  growth  ceases;  an  optimum  temperature,  at  which 
growth  is  most  luxuriant  and  rapid;  and  a  maximum  temperature, 
above  which  growth  ceases,  and  the  organisms  die.  Fischer1  has 
classified  bacteria  according  to  their  thermic  relations  as  follows: 

Minimum.      Optimum.      Maximum. 

1.  Psychrophilic  bacteria   .        0  15-20  30       Many  water  bacteria. 

2.  Mesophilic  bacteria        .     15-25  37  43       Pathogenic  bacteria  and 

others. 

3.  Thermophilic  bacteria   .    25-45  50-55  85       Spore-forming    bacteria 

from  soil,   feces,   and 
thermal   springs. 

Bacteria  which  are  progressively  pathogenic  for  man  and  warm- 
blooded animals  develop  within  a  much  narrower  range  of  tempera- 
ture than  the  saprophytic  bacteria  which  are  found  chiefly  in  nature, 
as  the  following  table,  also  taken  from  Fischer,2  indicates: 

Difference  between 


minimum  and 

Minimum. 

Optimum. 

Maximum. 

maximum. 

B.  phosphorescen 

s 

.        0 

20 

38 

38 

B.  fluorescens 

.        5 

20-25 

38 

33 

B.  subtilis 

.        6 

30 

50 

44 

Vibrio  cholerae 

.      10 

37 

40 

30 

B.  anthracis    . 

.      12 

37 

45 

33 

B.  diphtherise 

.     18 

33-37 

45 

27 

Mic.  gonorrhese 

.     25 

37 

39 

14 

B.  tuberculosis 

.     30 

37 

42 

12 

B.  thermophilus  . 

.     40 

60 

80 

40 

1  Vorlesungen  iiber  Bakterien,  1903,  II  Aufl. 

2  Loc.  cit.,  106. 


42  GENERAL  PHYSIOLOGY  OF  BACTERIA 

The  saprophytic  bacteria,  as  for  example  B.  subtilis,  which  develop 
through  a  relatively  wide  range  of  temperature  are  also  called 
Eurythermic  bacteria.  The  pathogenic  bacteria,  as  for  example  the 
tubercle  bacillus,  which  exhibit  but  little  latitude  in  this  respect,  are 
called  Stenothermic  bacteria. 

2.  Cold. — All  bacteria  grow  best  and  most  rapidly  in  an  environ- 
ment which  is  maintained  at  the  optimum  temperature  for  the  organism. 
If  this  temperature  is  lowered  even  a  few  degrees,  the  rate  of  reproduc- 
tion is  proportionately  reduced.  As  the  temperature  approaches  0°  C., 
there  is  complete  or  nearly  complete  cessation  of  growth  with  a  corre- 
sponding complete  or  nearly  complete  restriction  of  chemical  inter- 
change between  the  organism  and  its  environment.  The  viability, 
and  in  the  pathogenic  bacteria  the  virulence,  is  not  seriously  impaired 
even  by  exposure  to  these  low  temperatures  for  considerable  periods 
of  time.  Practical  advantage  is  taken  of  this  restriction  of  bacterial 
development  by  cold  in  the  preservation  of  food  by  refrigeration  or 
by  cold  storage,  and  also  for  the  preservation  of  laboratory  cultures 
of  many  non-spore-forming  bacteria  by  placing  them  in  the  ice-box 
at  5-10°  C.  So  resistant  are  bacteria  to  low  temperatures  that  they 
may  be  actually  frozen  solid  and  kept  in  this  state  for  days  and  even 
weeks  without  killing  all  the  individuals  of  the  cultures.  Alternate 
freezing  and  thawing  is  much  more  disastrous  to  them  than  simple 
freezing.  Thus,  typhoid  bacilli  may  be  suspended  in  water  and 
exposed  to  a  freezing  mixture  of  ice  and  salt  at  — 18°  C.  for  several 
weeks  without  killing  all  the  organisms,  although  the  majority  of  them 
are  killed  within  a  few  hours.  At  the  end  of  a  week  fully  90  per  cent, 
are  dead;  over  95  per  cent,  succumb  by  the  end  of  four  weeks'  continual 
freezing;  but  from  four  to  six  months'  continuous  freezing  is  required 
to  kill  all  of  the  typhoid  bacilli.  The  survivors  appear  to  be  no  more 
resistant  to  subsequent  freezing  than  similar  organisms  which  have 
not  been  frozen.  It  is  a  noteworthy  fact  that  bacteria  suspended  in 
colloidal  substances,  as  egg  albumen,  are  much  more  resistant  to  freez- 
ing than  similar  organisms  frozen  in  water.  Alternate  freezing  and 
thawing  in  colloids  is  much  less  disastrous  to  bacteria,  in  other  words, 
than  the  same  freezing  in  aqueous  solutions.  It  is  probable  that  the 
mechanical  factor  of  crystallization  which  takes  place  when  water  is 
frozen  actually  crushes  many  of  the  bacteria,  thus  accounting,  in  part 
at  least,  for  the  greater  death  rate  in  aqueous  solutions  than  that 
observed  in  colloids.  When  bacteria  are  once  frozen,  further  lowering 
of  the  temperature  has  surprisingly  little  influence  upon  the  death 
rate.  Typhoid  and  colon  bacilli  will  survive  freezing,  in  moderate 


HEAT  PRODUCTION  43 

numbers  at  least,  in  liquid  air  (  — 176°  C.)  or  even  liquid  hydrogen 
(—252°  C.)  for  several  hours,  and  develop  vigorously  when  they  are 
again  placed  in  a  suitable  environment  at  the  optimum  temperature. 

3.  Heat. — Bacteria  are  distinctly  injured  by  exposure  to  even  slight 
increases  of  temperature  above  that  optimum  for  their  growth,  although 
there  are  considerable  differences  met  with  among  different  kinds  of 
organisms  in  this  respect.  Generally  speaking,  the  saprophytic  bac- 
teria exhibit  greater  latitude  than  the  pathogenic  bacteria.  If  the 
maximum  temperature  of  growth  be  exceeded  by  even  a  very  few 
degrees,  the  death  of  the  organisms  follows  rather  promptly.  The 
greater  the  degree  of  heat,  the  shorter  the  time  required  to  kill  them. 
Therefore,  the  thermal  death  point  of  bacteria,  that  temperature  at 
which  specific  organisms  die,  is  dependent  not  only  upon  the  actual 
temperature  to  which  they  are  exposed,  but  also  to  the  length  of  time 
of  exposure.  A  standard  exposure  of  ten  minutes  has  been  proposed, 
so  that  the  thermal  death  point  of  the  bacterium  may  be  defined  as 
the  lowest  temperature  to  which  it  must  be  exposed  for  ten  minutes 
under  constant  conditions  to  ensure  the  sterility  of  the  culture.  The 
determination  of  the  thermal  death  point  is  influenced  by  many  factors 
besides  the  kind  of  organism  under  observation  and  the  temperature. 
Older  cultures  are  usually  less  resistant  than  younger  cultures  of  the 
same  kind.  The  reaction  of  the  medium  (acids  particularly  decrease 
thermal  resistance),  the  presence  of  extraneous  substances  as  mucin 
and  other  non-conductors  of  heat,  all  play  a  part.  Certain  modifica- 
tions in  the  characteristics  of  bacteria  are  observed  when  they  are 
exposed  for  several  hours  at  the  maximum  temperature  of  growth 
or  a  degree  or  ,two  above  this  point.  For  example,  anthrax  bacilli, 
which  habituallyjorm  spores,  lose  this  property  when  they  are  exposed 
to  44°  C.  for  several  hours. 

Dry  Heat,  Moist  Heat — Dry  heat  is  less  effective  in  killing  bacteria 
than  moist  heat.  This  is  shown  by  the  high  temperature  to  which 
glassware  and  other  apparatus  must  be  exposed  in  order  to  kill  spores, 
a  temperature  of  160°  C.  for  one  and  one-half  hours  being  required 
to  ensure  sterility.  Moist  heat,  which  is  best  obtained  by  dry  steam 
under  pressure,  will  kill  even  the  most  resistant  spores  in  fifteen 
minutes  at  fifteen  pounds  pressure. 

H.     HEAT  PRODUCTION.  . 

The  energy  liberated  by  bacteria  during  the  decomposition  of  organic 
substances  by  bacterial  growth  is  partly  utilized  by  them  for  their 


44  GENERAL  PHYSIOLOGY  OF  BACTERIA 

anabolic  requirements.  A  larger  part,  however,  is  dissipated  as  heat. 
The  heat  generated  in  actively  growing  cultures  of  bacteria  can  be 
detected  with  sensitive  thermometers,  provided  losses  due  to  radiation 
and  evaporation  are  guarded  against.  The  heat  production  is  not 
great  as  a  rule,  although  in  certain  fermentations  it  may  rise  as  high 
as  12-15°  above  the  uninoculated  controls.  The  decomposition  of 
protein  and  protein  derivatives  (putrefaction)  usually  gives  rise  to  less 
heat  than  the  decomposition  of  carbohydrates  (fermentation)  under 
the  same  conditions. 

I.     LIGHT   AND   ELECTRICITY. 

The  vast  majority  of  plants  possess  a  photodynamic  pigment, 
chlorophyll.  This  pigment  can  synthesize  inorganic  substances,  as 
CO2  and  water,  together  with  nitrates,  into  complex  organic  compounds 
through  the  energy  of  the  sun's  rays  acting  upon  it.  Plants  possessed 
of  this  pigment,  therefore,  are  the  synthetic  agents  of  nature.  Usually 
this  pigment  is  green;  it  may,  however,  be  brown  or  red,  the  latter 
pigment  being  characteristic  of  certain  algaB.  A  group  of  the  higher 
bacteria,  the  Rhodobacteriaceae,  possess  a  photodynamic  pigment, 
bacteriopurpurin,  which  appears  to  be  analogous  to  chlorophyll  of 
the  green  plants.  These  sulphur  bacteria  prefer  light  and  move  toward 
it.1  The  action  of  sunlight  on  this  bacteriopurpurin  enables  them  to 
decompose  CO2  and  to  utilize  the  oxygen  thus  obtained  to  oxidize 
H2S. 

All  other  known  bacteria  have  no  photodynamic  pigment.  Light 
is  not  a  source  of  energy  to  them,  and  they  are  distinctly  harmed  by 
it;  they  grow  best  in  darkness.  Direct  daylight  kills  them  rapidly, 
and  even  prolonged  exposure  to  diffuse  light  may  be  fatal.  Bacteria 
are  more  rapidly  killed  by  exposure  to  the  sun's  rays  in  June,  July 
and  August2  than  exposure  of  the  same  time  in  November,  December 
and  January.  Expressed  differently,  many  bacteria  which  are  killed 
after  an  exposure  of  from  one  to  two  hours'  direct  sunlight  in  summer 
require  an  exposure  of  from  two  to  three  hours  in  winter  to  accomplish 
the  same  result. 

Of  the  spectral  rays,  the  red  and  infra-red  rays,  aside  from  the  heating 
effect,  are  without  noteworthy  action  on  bacteria.  The  blue,  violet, 
and  ultraviolet  rays,  on  the  contrary,  are  distinctly  bactericidal. 

1  Yost,  Plant  Physiology,  223. 

2  In  the  Northern  Hemisphere. 


LIGHT  AND  ELECTRICITY  45 

These  rays  are  chemodynamic  and  it  is  very  probable  that  the  death 
of  bacteria  exposed  to  them  in  organic  media  results  from  the  formation 
of  H2O2  or  other  germicidal  substances  from  the  substrate.  Bacteria 
are  also  killed  in  non-decomposable  media  when  they  are  exposed  to 
the  ultraviolet  rays.  It  should  be  remembered  that  one  of  the  most 
important  characteristics  of  ultra  spectral  emanations  is  their  very 
short  wave  length.  Glass  is  opaque  to  them  where  quartz  is  trans- 
parent. 

Electricity. — It  is  difficult  to  differentiate  sharply  between  purely 
electrical  effects  and  chemical  changes  which  are  induced  in  media 
of  various  kinds  by  the  action  of  electric  currents.  Generally  speaking, 
strong  electrical  currents  sterilize  media  in  which  bacteria  are  growing, 
but  it  is  by  no  means  certain  that  the  electric  current  per  se  is  the 
important  factor.  Zeit1  has  made  a  careful,  extensive  and  accurate 
study  of  the  action  of  various  kinds  of  electric  currents  on  bacterial 
growth,  and  his  conclusions  are  as  follows: 

"LA  continuous  current  of  260  to  320  milliamperes  passed  through 
bouillon  cultures  kills  bacteria  of  low  thermal  death  points,  in  ten 
minutes  by  the  production  of  heat — 98.5  °  C.  The  antiseptics  produced 
by  electrolysis  during  this  time  are  not  sufficient  to  prevent  growth  of 
even  non-spore-bearing  bacteria.  The  effect  is  a  purely  physical  one. 

"  2.  A  continuous  current  of  48  milliamperes  passed  through  bouillon 
cultures  for  from  two  to  three  hours  does  not  kill  even  non-resistant 
forms  of  bacteria.  The  temperature  produced  by  such  a  current  does 
not  rise  above  37°  C.  and  the  electrolytic  products  are  antiseptic  but 
not  germicidal. 

"  3.  A  continuous  current  of  100  milliamperes  passed  through  bouillon 
cultures  for  seventy-five  minutes  kills  all  non-resistant  forms  of  bac- 
teria even  if  the  temperature  is  artificially  kept  below  37°  C.  The 
effect  is  due  to  the  formation  of  germicidal  electrolytic  products  in  the 
culture.  Anthrax  spores  are  killed  in  two  hours.  Subtilis  spores  were 
still  alive  after  the  current  was  passed  for  three  hours. 

"4.  A  continuous  current  passed  through  bouillon  cultures  of  bacteria 
produces  a  strongly  acid  reaction  at  the  positive  pole,  due  to  the  libera- 
tion of  chlorin  which  combines  with  oxygen  to  form  hypochlorous  acid. 
The  strongly  alkaline  reaction  of  the  bouillon  culture  at  the  negative 
pole  is  due  to  the  formation  of  sodium  hydroxid  and  the  liberation  of 
hydrogen  in  gas  bubbles.  With  a  current  of  100  milliamperes  for  two 
hours  it  required  8.82  milligrams  of  H2SO4  to  neutralize  1  c.c.  of  the 

1  Jour.  Am.  Med.  Assn.,  November,  1901. 


46  GENERAL  PHYSIOLOGY  OF  BACTERIA 

culture  fluid  at  the  negative  pole,  and  all  the  most  resistant  forms  of 
bacteria  were  destroyed  at  the  positive  pole,  including  anthrax  and 
subtilis  spores.  At  the  negative  pole  anthrax  spores  were  killed  also, 
but  subtilis  spores  remained  alive  for  four  hours. 

"  5.  The  continuous  current  alone,  by  means  of  DuBois-Reymond's 
method  of  non-polarizing  electrodes  and  exclusion  of  chemical  effects 
by  ions  in  Kruger's  sense,  is  neither  bactericidal  nor  antiseptic.  The 
apparent  antiseptic  effect  on  suspensions  of  bacteria  is  due  to  electric 
osmose.  The  continuous  electric  current  has  no  bactericidal  nor 
antiseptic  properties,  but  can  destroy  bacteria  only  by  its  physical 
effects — heat — or  chemical  effects,  the  production  of  bactericidal 
substances  by  electrolysis. 

"  6.  A  magnetic  field,  either  within  a  helix  of  wire  or  between  the 
poles  of  a  powerful  electro-magnet,  has  no  antiseptic  or  bactericidal 
effects  whatever. 

"7.  Alternating  currents  of  a  three-inch  Ruhmkorff  coil  passed 
through  bouillon  cultures  for  ten  hours  favor  growth  and  pigment 
production. 

"8.  High  frequency,  high  potential  currents — Tesla  currents — have 
neither  antiseptic  nor  bactericidal  properties  when  passed  around  a 
bacterial  suspension  within  a  solenoid.  When  exposed  to  the  brush 
discharges,  ozone  is  produced  and  kills  the  bacteria. 

"9.  Bouillon  and  hydrocele-fluid  cultures  in  test-tubes  of  non- 
resistant  forms  of  bacteria  could  not  be  killed  by  Rontgen  rays  after 
forty-eight  hours'  exposure  at  a  distance  of  20  mm.  from  the  tube. 

"  10.  Suspensions  of  bacteria  in  agar  plates  and  exposed  for  four 
hours  to  the  rays,  according  to  Rieder's  plan,  were  not  killed. 

"11.  Tubercular  sputum  exposeu  to  the  Rontgen  rays  for  six  hours 
at  a  distance  of  20  mm.  from  the  tube,  caused  acute  miliary  tubercu- 
losis of  all  the  guinea-pigs  inoculated  with  it. 

"  12.  Rontgen  rays  have  no  direct  bactericidal  properties.  The 
clinical  results  must  be  explained  by  other  factors,  possibly  the  pro- 
duction of  ozone,  hypochlorous  acid,  extensive  necrosis  of  the  deeper 
layers  of  the  skin,  and  phagocytosis." 

J.     GRAVITY,  OSMOTIC  PRESSURE,  AGITATION,  CHEMOTAXIS. 

1.  Gravity. — The  majority  of  bacteria  suspended  in  liquids  are 
not  killed  even  by  four  hours'  exposure  to  direct  pressure  of  from  2000 
to  3000  atmospheres  (one  atmosphere  of  pressure  is  equal  to  approxi- 


GRAVITY,  OSMOTIC  PRESSURE,  AGITATION,  CHEMOTAXIS       47 

mately  15  pounds  to  the  square  inch,  or  one  kilogram  per  square 
centimeter  of  surface).  Bacteria  are  weakened,  however,  by  these 
great  pressures,  as  is  evidenced  by  a  diminution  in  virulence,  decreased 
pigment  production,  and  the  partial  or  complete  inability  to  multiply. 
It  is  a  curious  fact  that  motile  bacteria  may  retain  their  motility  after 
an  exposure  of  several  hours  to  2000  atmospheres  from  the  pressure 
liquids,  even  although  their  powers  of  reproduction  are  quite  lost. 

Liquids  are  practically  non-compressible,  consequently  direct  pres- 
sure does  not  affect  the  volume  of  the  liquid  in  which  bacteria  are 
suspended,  nor  does  this  pressure  affect  the  amount  of  gas  dissolved 
in  the  liquid.  If,  however,  bacteria  are  exposed  in  liquids  to  gas 
pressure  in  the  place  of  direct  pressure,  the  germicidal  action  of  the 
gas  plays  the  prominent  part  in  the  final  result.  The  amount  of  gas 
dissolved  in  the  liquid  increases  with  increase  of  pressure,  consequently 
feebly  germicidal  gases  may  become  powerfully  germicidal  as  the 
pressure  is  increased.  Thus,  bacteria  suspended  in  water  overlaid 
by  CO2,  which  is  feebly  germicidal  at  ordinary  pressures,  are  rapidly 
killed  if  the  pressure  is  gradually  increased;  that  is,  CO2  under  these 
conditions  becomes  strongly  bactericidal.  According  to  Certes,1 
600  atmospheres  pressure  of  an  inert  gas,  as  nitrogen,  will  not  kill 
anthrax  bacilli. 

Diminished  Pressure. — Diminished  pressure,  aside  from  lowering 
the  oxygen  tension  to  a  point  below  that  necessary  for  the  growth  of 
aerobic  bacteria,  does  not  interfere  seriously  with  bacterial  growth. 

2.  Osmotic  Pressure. — The  boundary  layer,  ectoplasm,  of  every 
bacterial  cell  reacts  like  a  semi-permeable  or  osmotic  membrane. 
Through  this  membrane  must  pass  all  the  elements  necessary  to  the 
nutrition  of  the  organism.  A  normal  bacterial  cell  always  tends  to 
maintain  a  greater  concentration  of  solutes  within  its  substance  than 
exists  in  the  surrounding  medium;  hence  the  pressure  from  within 
upon  the  cell  membrane  is  somewhat  greater  than  the  pressure  from 
without  upon  the  cell  membrane,  and  the  cell  is  consequently  in  a 
state  of  continual  turgor.  The  osmotic  pressure  exerted  by  dissolved 
substances  varies  very  greatly.  Those  of  high  molecular  weight,  as 
albuminoses  or  peptones,  exert  little  or  no  osmotic  pressure.  Crys- 
talloids, on  the  contrary,  may  exert  very  considerable  pressure.  Thus, 
a  30  per  cent,  solution  of  dextrose  exerts  a  pressure  of  about  22  atmos- 
pheres. A  bacterial  cell  placed  in  such  a  solution  is  under  a  great 
strain.  If  bacteria  which  are  in  a  state  of  equilibrium  with  reference 

1  Compt.  rend.  Acad.  de  sc.,  1884,  99,  385. 


48  GENERAL  PHYSIOLOGY  OF  BACTERIA 

to  the  osmotic  pressure  of  a  solution  are  suddenly  introduced  into 
media  containing  a  greater  concentration  of  solutes,  the  contents  of 
the  cell  diminish  somewhat  in  amount,  due  to  the  rapid  withdrawal  of 
water  leaving  the  rigid  cell  membrane  visible.  This  shrinkage  of  the 
cell  contents  is  spoken  of  as  plasmolysis.1  This  shrinkage  of  the  cell 
contents  would  indicate  that  the  cell  membrane  is  differentially  more 
rapidly  permeable  to  water  than  to  crystalloids.  All  bacteria  are  not 
plasmolyzed  when  they  are  suddenly  introduced  into  hypertonic 
solutions,  and  some  organisms  exhibit  the  phenomenon  of  plasmo- 
lysis to  a  much  greater  extent  than  others.  Plasmolysis  does  not  neces- 
sarily result  in  the  death  of  the  organism.  It  appears  to  be  a  fact  that 
older  bacteria  are  frequently  more  readily  plasmolyzed  than  younger 
individuals  of  the  same  kind.  The  observations  of  Nicolle  and  Auclaire2 
would  indicate  that  bacteria  which  retain  the  Gram  stain  are  less 
readily  plasmolyzed  than  Gram-negative  bacteria.  » Whether  Gram- 
positive  bacteria  which  have  become  Gram-negative  due  to  prolonged 
cultivation  in  artificial  media  invariably  follow  the  same  rule  is  not 
known. 

If  bacteria  are  gradually  subjected  to  solutions  of  greater  or  lesser 
osmotic  pressure,  they  usually  accommodate  themselves  to  these 
changes  without  visible  effect.  If  bacteria  are  introduced  abruptly 
into  solutions  of  low  osmotic  pressure  or  distilled  water,  water  rapidly 
passes  through  the  cell  membrane  of  the  bacteria  faster  than  the  solutes 
within  the  cell  can  pass  out,  thus  rapidly  increasing  the  intracellular 
pressure  until  frequently  the  cell  membrane  ruptures,  permitting  the 
escape  of  some  of  the  cell  contents.  This  phenomenon  is  called 
plasmoptysis.3  Most  bacteria  do  not  plasmoptyze  readily,  and  it  is 
problematical  how  much  importance  should  be  attached  to  either 
plasmolysis  or  plasmoptysis  in  practical  bacteriology. 

3.  Agitation. — Bacteria  grow  best  in  quiet  surroundings,  although 
a  slight  amount  of  agitation  is  usually  harmless  and  may  be  even 
beneficial  if  it  tends  to  dislodge  waste  products  from  the  immediate 
surroundings  of  sedimented  organisms.  Rapid  agitation  frequently 
retards  the  multiplication  of  bacteria  in  fluid  cultures,  and  Meltzer4 
and  Horvath5  have  shown  that  violent  shaking  gradually  kills  bacteria; 
not,  however,  by  rupturing  the  cell  membrane.  The  organisms  undergo 

1  Fischer,  loc.  cit.,  p.  23. 

2  Ann.  de  1'Inst.  Pasteur,  1909,  xxiii,  547. 

3  Fischer,  loc.  cit.,  p.  48. 

4  Ztschr.  f.  Biol.,  1894,  xxx. 
6  Pfliiger's  Arch.,  1887,  xvii. 


ENZYMES,   TOXINS,  PTOMAINS  49 

a  gradual  disintegration,  and  the  injurious  effects  observed  are  said 
by  these  observers  to  be  not  purely  mechanical. 

4.  Chemotaxis. — Bacteria  respond  to  various  chemical  stimuli. 
Substances  which  can  be  used  by  them  for  nutritional  purposes,  as 
various  constituents  of  laboratory  media,  appear  to  attract  bacteria. 
Harmful  substances,  as  acids  or  alkalis,  may  act  in  the  reverse  manner. 
Oxygen  is  a  powerful  chemotactic  agent  for  many  aerobic  bacteria, 
while  many  anaerobes  are  repelled  by  it.  The  mutual  chemotactic 
relations  of  bacteria  and  leukocytes,  and  the  well-defined  tendency 
of  certain  invasive  bacteria  to  localize  in  definite  tissues  or  organs 
of  the  animal  body  .are  interesting  fields  for  speculation.  Nothing 
conclusive  is  known  about  these  relations. 

K.     ENZYMES,  TOXINS,  PTOMAINS. 

Enzymes. — The  phenomena  of  chemical  interchange  between 
bacteria  and  their  environment  indicate  that  enzyme  activity  plays 
an  important  part  in  bacterial  metabolism. 

Enzymes  may  be  defined  as  substances  of  unknown  composition 
produced  by  living  cells  which  incite  specific  chemical  reactions  with- 
out permanently  combining  with  the  products  of  reactien.  A  small 
amount  of  enzyme  acting  under  favorable  conditions  will  cause  a 
relatively  extensive  transformation  of  substance  without  itself  being 
used  up  or  inactivated.  There  is,  however,  a  limit  to  the  amount  of 
transformation  which  a  given  amount  of  enzyme  can  accomplish, 
for  the  accumulation  of  reaction  products  tends  to  restrict  enzyme 
action;  the  removal  of  reaction  products  appears  to  extend  enzyme 
action  somewhat.  All  bacterial  cells  appear  to  produce  or  to  possess 
enzymes,  probably  several,  which  may  be  divided  somewhat  arbi- 
trarily into  two  classes,  the  extracellular  or  exo-enzymes,  and  the 
intracellular  or  endo-enzymes. 

Exo-enzymes. — Exo-enzymes  are  those  which  are  excreted  from  the 
organism  and  appear  as  soluble,  filterable  and,  frequently,  diffusible 
enzymes,  which  may  be  obtained  in  an  active  state  from  filtrates  of 
cultures  of  bacteria.  Their  diffusion  from  the  bacterial  cell  and  their 
filterability  suggests  that  they  may  be  relatively  simple  in  molecular 
aggregation.  Their  function  is  essentially  a  "preparatory"  one,  for 
they  transform  potential  nutritional  substances,  as  proteins,  carbo- 
hydrates or  fats,  to  simpler  compounds  which  are  assimilable  by  the 
bacteria.  It  is  very  probable  that  the  exe-enzymes  work  uneconem- 


50  GENERAL  PHYSIOLOGY  OF  BACTERIA 

ically  in  the  sense  that  they  transform  more  material  than  the 
organisms  require :  this  phenomenon  is  exhibited  in  the  extensive  lique- 
faction of  gelatin  by  proteolytic  bacteria,  as  B.  proteus.  The  organism^ 
which  elaborates  such  an  exo-enzyme  probably  derives  but  little  energy 
from  its  activity,  and,  conversely,  probably  expends  comparatively 
little  energy  in  the  elaboration  and  secretion  of  the  exo-enzyme. 

Endo-enzymes. — Comparatively  little  is  known  of  the  endo-enzymes : 
it  is -generally  believed  that  they  are  comparatively  non-diffusible,  at 
least  in  an  active  state,  and  that  they  are  non-  or  but  slightly  filter- 
able. This  suggests  that  they  are  relatively  complex  in  their  mole- 
cular aggregation.  Their  function  is  probably  to  act  upon  the  nutrient 
substances  which  the  cell  has  assimilated,  partly  to  liberate  energy 
from  them,  and  partly  to  participate  in  the  organization  of  the  cell 
constituents.  These  endo-enzymes  work  economically  in  contra- 
distinction to  the  exo-enzymes  in  the  sense  that  the  substrate  is  appar- 
ently changed  by  them  in  proportion  to  the  requirements  of  the  cell. 
Endo-enzymes  may  be  obtained  from  bacterial  cells  when  the  latter 
disintegrate,  provided  the  rupture  of  the  cells  is  not  accomplished  by 
violent  chemical  means.  Probably  the  phenomena  of  autolysis  which 
many  bacteria  exhibit  when  they  are  placed  in  an  environment  free 
from  food  may  be  due,  in  part  at  least,  to  the  autodigestion  of  the 
organisms  by  their  endo-enzymes. 

Classification  of  Enzymes. — Enzymes  are  usually  classified  according 
to  the  substrate  they  act  upon :  thus,  proteolytic  enzymes,  or  proteases, 
split  proteins  or  protein  derivatives  into  simpler  compounds;  carbo- 
hydrolytic  enzymes  split  starches  or  polysaccharides  into  simpler 
carbohydrates;  fat-splitting  ferments,  lipases,  split  fats  into  glycerin 
and  fatty  acids.  The  above  enzymes  are  -hydrolytic  in  character, 
that  is,  they  effect  cleavage  of  protein  or  carbohydrate  or  fat  or  of 
glucosides  by  splitting  the  molecule  into  simpler  molecules  which 
simultaneously  take  up  hydrogen  and  oxygen  in  the  proportions  to 

form  water,  thus: 

i. 

CH2NH2CO-NHCH2COOH  +  H2O  (+  enzyme)  =  CH2NH2COOH  +  CH2NH2COOH. 
Glycyl-glycine.  Glycine.  Glycine. 

2. 

Ci2H22On  +  H2O  ( +  lactase)  =  C6Hi2O6  +  C6Hi2O6. 
Lactose.  Dextrose.       Galactose. 

3. 

CH2O-CO-CH3  CH2OH 

I  I 

CHO-CO-CHs    +  3  H2O  (+  lipase)  =  CHOH   +  3  CH3COOH 

Acetic  acid. 

CH2OTCO7CH3  CH2OH 

Triacetin,  Glycerin. 


ENZYMES,   TOXINS,  PTOMAINS  51 

The  question  of  specificity  of  action  of  bacterial  enzymes  is  not 
definitely  settled.  There  is  some  evidence  in  favor  of  the  view  that 
exo-proteolytic  enzymes  produced  by  various  bacteria  act  upon  a 
variety  of  proteins:  thus,  the  cholera  vibrio  produces  a  soluble  pro- 
teolytic  enzyme  which  will  digest  casein,  coagulated  blood  serum,  egg 
albumen,  fibrin  and  gelatin.  Other  organisms,  as  the  staphylococcus, 
produce  an  exo-enzyme  which  will  hydrolyze  casein,  coagulated  blood 
serum  and  gelatin:  its  action  upon  other  proteins  is  not  definitely 
established.  The  important  question — are  the  products  of  hydrolysis 
of  the  same  protein  by  proteolytic  enzymes  from  different  bacteria 
the  same — is  not  definitely  settled;  it  is  probable,  however,  that  the 
products  differ.  This  suggests  that  the  proteolytic  enzymes  of  bacteria 
are  not  mere  "catalyzers"  which  accelerate  reactions  in  relatively 
unstable  substances  that  would  take  place  spontaneously  but  much 
more  slowly;  these  enzymes  (proteolytic  enzymes)  may  not  only  incite 
reaction,  they  may  guide  it,  as  it  were,  along  lines  of  cleavage  which 
would  not  be  followed  in  the  absence  of  this  enzyme.  The  carbo- 
hydrate— and  the  fat-splitting  enzymes  have  much  less  latitude  in 
splitting  the  carbohydrates  and  fats  respectively  than  the  proteolytic 
enzymes,  for  these  substances  are  less  complex  in  structure  and  com- 
position than  the  proteins  and  protein  derivatives. 

Fuhrmann1  has  classified  enzymes  of  bacterial  origin  into  four  types 
as  follows: 

A.  SCHIZASES  (HYDROLYTIC)  CLEAVAGE  ENZYMES. 

1.  Proteases,  protein-splitting  enzymes.      Pepsin,  Trypsin  (Lysins,  Coagu- 


2.  Carbohydrate-splitting  enzymes.    Amylase,  Cellulase,  Pectinase,  Gelase, 

Invertase,  Lactase. 

3.  Glucoside-splitting  enzymes.     Emulsin  (Synaptase). 

4.  Fat-splitting  enzymes.      Lipases  (esterases). 

B.  OXIDIZING  ENZYMES. 

Tyrosinase,  Acetic  bacteria,  Oxydase. 

C.  REDUCING  ENZYMES. 

Reductases. 

D.  FERMENTATION  ENZYMES. 

Zymase,  Urease,  Lactic  acid  enzyme. 

The  bacteriolysins  are  of  particular  importance  in  bacteriology: 
of  the  bacteriolysins,  those  which  liberate  unchanged  hemoglobin 
from  red  blood  cells  (hemolysins)  and  those  which  digest  hemoglobin 
(hemodigestins2)  are  intermediary  in  their  general  properties  between 
enzymes  and  toxins,  if  indeed  there  is  any  tangible  distinction  between 

1  Vorlesungen  uber  Bakterienenzyme,  Jena,  1907. 

2  Van  Loghem,  Centralbl.  f.  Bakteriol.,  1912-1913,  Ixvii,  410. 


52  GENERAL  PHYSIOLOGY  OF  BACTERIA 

them.  Vaughan1  has  studied  both  enzymes  and  toxins  extensively, 
and  has  summarized  admirably  the  points  of  resemblance  between 
exo-enzymes  and  exo-toxins  as  follows: 

"1.  Both  are  destroyed  by  heat.2 

"2.  They  act  in  very  dilute  solution. 

"3.  When  repeatedly  injected  into  animals  in  non-fatal  doses  they 
cause  the  body  cells  to  elaborate  antibodies  which  neutralize  the  toxin 
(or  the  enzyme)  both  in  viw  and  in  vitro. 

"4.  In  the  development  of  their  effects  a  period  of  incubation  is 
required. 

"5.  It  has  been  shown  (by  Abderhalden)  by  optical  methods  that 
they  have  a  cleavage  effect  upon  proteins — they  split  complex  proteins 
into  simpler  bodies;  in  other  words,  they  have  a  proteolytic  action. 

"6.  They  are  specific  in  two  senses:  (a)  they  are  specific  according 
to  the  cell  which  produces  them;  (6)  they  are  specific  in  the  antibody 
elaborated  in  the  animal  body  after  repeated  injections  of  non-fatal 
doses." 

Bacterial  toxins  are  usually  classified  as  exo-  or  soluble  (extra- 
cellular) toxins,  and  endo-  (intracellular)  toxins.  The  former  are 
soluble  and  diffuse  out  from  the  bacterial  cell  into  the  surrounding 
medium.  Very  few  bacteria  produce  exo-toxins:  the  best  known  are 
those  of  the  diphtheria,  tetanus,  and  botulismus  bacilli.  To  these 
specific  antitoxins  are  known.  Endo-toxins  are  non-diffusible  and  are 
locked  up  in  the  bacterial  cell;  they  are  liberated  only  when  the 
cell  disintegrates.  No  specific  antitoxin  has  been  produced  for  an 
endo-toxin. 

Ptomains. — Ptomains  are  soluble,  basic,  nitrogen-containing  sub- 
stances formed  from  proteins  or  protein  derivatives  by  the  action  of 
microorganisms.  They  are  non-specific,  relatively  poor  in  oxygen 
content,  and  probably  simpler  in  composition  than  either  exo-  or  endo- 
toxins.  No  antibodies  have  been  produced  against  them.  Some  are 

poisonous,  many  are  not. 

\ 
\ 

L.     PIGMENTS. 

With  the  exception  of  bacteriopurpurin,  which  occurs  in  the  sulphur 
bacteria  and  is  supposed  to  be  photodynamic  and,  therefore,  somewhat 
analogous  to  the  chlorophyll  of  the  higher  plants,  the  significance  of 

1  Protein  Split  Products,  Lea  &  Febiger,  Philadelphia  and  New  York,  1913. 

2  Although  they  are  somewhat  more  resistant  to  heat  than  the  cells  which  produce 
them. 


SYMBIOSIS,  ANTIBIOSIS  AND  COMMENSALISM  53 

pigment  formation,  which  is  a  striking  cultural  characteristic  of  many 
bacteria,  is  wholly  unknown.  The  pigment  they  produce  does  not 
protect  them 'against  strong  Light,  and  achromogenic  strains  may  be 
cultivated  from  the  chromogenic  varieties  without  apparent  loss  in  the 
cultural  or  chemical  characters  of  the  organisms.  It  is  very  probable 
that  these  pigments  are  chiefly  waste  products  of  metabolic  origin. 

Pigments  are  produced  in  darkness  and  sunlight  rapidly  destroys 
many  of  them.  Oxygen  is  not  necessary  for  their  production,  for  the 
non-colored  leukobase  is  the  form  in  which  the  pigment  is  excreted 
by  bacteria,  but  oxygen  is  necessary  for  the  development  of  color  from 
this  leukobase. 

Pigment-producing  bacteria  may  be  grouped  into  four  classes : 

1.  Bacteria  producing  photodynamic  pigment.     Certain   sulphur 
bacteria  which  produce  bacteriopurpurin. 

2.  Phosphorogenic  bacteria  which  produce  a  luminous  substance 
somewhat  analogous  to  that  of  glow-worms.     These  organisms  are 
chiefly  marine  forms,  as  B.  phosphorescens. 

3.  Fluorogenic  bacteria  which  produce  a  pigment  soluble  in  water 
and  culture  media;  this  usually  exhibits  complementary  colors  as  it 
is  viewed  by  reflected  and  transverse  light  respectively. 

4.  Chromogenic  bacteria.    The  pigment  produced  is  usually  insol- 
uble in  water  and  soluble  in  organic  solvents.    The  color  varies  accord- 
ing to  the  organism  producing  it.    The  more  common  colors  are  red, 
orange,  yellow,  green,  blue,  violet,  brown,  and  black  pigment.    These 
colored  pigments  are  usually  referred  to  as  lipochromes  because  of 
their  solubility  in  organic  solvents  and  their  general  relationship  to 
fats.    Many  of  them  give  well-defined  and  constant  absorption  when 
they  are  viewed  spectroscopically  in  solutions.1 

M.     SYMBIOSIS,   ANTIBIOSIS   AND   COMMENSALISM. 

The  biological  relations  of  bacteria  are  of  the  greatest  importance 
in  the  economy  of  nature  and  in  the  production  of  disease.  Bacteria 
do  not  grow  in  pure  culture  in  nature,  although  they  may  do  so  in  the 
tissues  of  man  or  animals,  as  disease-producing  bacteria  (pathogenic 
bacteria).  In  nature,  where  the  reduction  of  dead  complex  organic 
material  to  mineralized  salts  is  the  striking  function  of  bacteria,  the 
successive  steps  in  the  degradation  of  organic  matter  are  carried  on 
by  different  kinds  of  microbes.  The  various  steps  appear  to  vary 

1  Sullivan,  Jour.  Med.  Research,  1905,  xiv,  109. 


54  GENERAL  PHYSIOLOGY  OF  BACTERIA 

somewhat,  but  the  process  is  on  the  whole  an  orderly  and  definite  one. 
The  association  of  various  kinds  of  bacteria  in  this  process,  where 
each  succeeding  kind  profits  by  the  activities  of  the  preceding  kind,* 
is  a  symbiotic  one;  that  is,  the  several  types  of  organisms  mutually 
profit  by  their  combined  activities. 

It  frequently  happens  that  the  products  of  symbiotic  activity  may 
be  greater  than  the  sum  of  the  products  of  the  separate  activities  of 
the  organisms.1  On  the  contrary,  many  instances  are  known  in  which 
one  kind  of  organism  by  its  activity  actually  crowds  out  a  preexisting 
organism,  as  for  example,  the  lactic  acid  bacteria  which  sour  milk. 
They  produce  sufficient  lactic  acid  from  the  fermentation  of  the  lactose 
to  kill  the  proteolytic  forms.  This  substitution  of  one  type  of  organism 
by  another  is  known  as  antibiosis:  the  latter  organism  profits  wholly 
at  the  expense  of  the  first  organism. 

It  not  infrequently  happens  that  one  type  of  bacterium  profits  by 
the  activity  of  another  type  of  organism  without  benefiting  the  former 
in  return.  If  two  types  of  bacteria  are  concerned,  the  process  is  known 
as  metabiosis;  if  the  bacterium  is  living  on  a  host,  the  relationship  is 
spoken  of  as  parasitism. 

N.    MEDIA— COMPOSITION  AND  REACTION. 

Most  bacteria  grow  best  in  a  medium  containing  a  large  percentage 
of  moisture  in  which  diffusible  proteins  or  protein  derivatives  are 
present  as  sources  of  nitrogen:  these  substances  are  better  adapted  to 
the  dietary  needs  of  the  majority  of  bacteria  than  are  ammonium 
salts  or  even  simple  amino  acids.  A  very  few  bacteria  (nitrifying 
bacteria)  cannot  grow  in  media  containing  organic  nitrogen  compounds : 
a  few  strictly  pathogenic  bacteria  appear  to  require  nitrogen  as  it 
exists  in  the  highly  complex  tissues  of  man  or  animals  for  their  growth. 
Many  bacteria  can  utilize  carbohydrates  for  their  carbon,  hydrogen, 
and  oxygen  requirements.  Some  bacteria  appear  to  be  able  to  utilize 
fats  for  their  carbon  requirement. 

A  neutral  or  feebly  alkaline  reaction  is  best  adapted  to  the  develop- 
ment of  the  vast  majority  of  bacteria;  a  few  types  develop  best  in  a 
medium  which  is  distinctly  acid — the  aciduric  bacteria.2  Mineral 
acids  are  germicidal;  organic  acids  may  be  utilized  by  bacteria  for 
foods. 

1  Kendall,  Jour.  Am.  Med.  Assn..  1911,  Ivi,  1084. 

2  Kendall,  Jour.  Med.  Research,  1910,  xxii,  153. 


GROWTH  OF  BACTERIA  IN  THE  ANIMAL  BODY  55 

O.      GROWTH   OF   BACTERIA   IN   THE   ANIMAL  BODY. 

The  vast  majority  of  bacteria  do  not  grow  in  the  tissues  of  the  body, 
although  a  small  number  of  organisms,  the  parasitic  bacteria,  live 
habitually  on  the  surface  of  the  body  or  on  mucous  membranes,  usually 
without  producing  noticeable  effects.  A  small,  formidable  group  of 
bacteria,  the  progressively  pathogenic  bacteria,  actually  invade  the 
tissues;  they  may  produce  within  the  host  inhibition  of  function  or 
anatomical  changes  incompatible  with  health. 


CHAPTER  III. 


THE  CHEMISTRY  OF  BACTERIA.    THE  EFFECT  OF 
BACTERIA  ON  THEIR  ENVIRONMENT. 


A.  GENEEAL. 

B.  CHEMICAL  CONSTITUTION   OF   BAC- 

TERIA. 

1.  Elementary  Composition. 

2.  Chemical  Constitution. 

3.  Chemical  Composition. 

C.  COMPOSITION  OF  THE  MORPHOLOGI- 

CAL   COMPONENTS    OF    THE 
BACTERIAL  CELL. 

1.  Cell  membrane. 

2.  Capsule. 


3.  Cytoplasm. 

4.  Spores. 

D.  FOOD  RELATIONSHIPS  OF  BACTERIA. 

1.  General. 

2.  Sources  of  Food. 

(a)  Nitrogen. 
(&)  Carbon. 

(c)  Hydrogen. 

(d)  Oxygen. 

(e)  Inorganic  Salts. 


A.     GENERAL   CHEMISTRY   OF   BACTERIA. 

THE  practical  significance  of  bacteria  is  summed  up  in  the  nature 
and  extent  of  the  chemical  changes  which  they  induce  in  their  environ- 
ment, the  result  of  their  multiplication  and  vegetative  activity.  These 
changes  are  essentially  analytical,  for  the  function  of  bacteria  in 
nature  is  to  transform  dead  organic  matter  from  complex  unstable 
combinations  of  carbon,  hydrogen,  nitrogen,  and  oxygen,  which  are 
worthjess  in  the  economy  of  nature,  to  fully  mineralized,  stable  inor- 
ganic compounds  of  these  elements,  which  may  be  resynthesized  by 
plants. 

A  small  but  formidable  group  of  bacteria,  chiefly  those  pathogenic 
for  plants,  animals  and  man,  act  directly  upon  the  living  plant  or 
animal  organism,  producing  changes  in  them  which  may  be  tempor- 
arily incompatible  with  their  well-being,  and  not  infrequently  lead  to 
their  death  and  eventually  to  their  mineralization.  The  pathogenic 
bacteria,  therefore,  are  also  analytical  in  their  activities  and  do  not 
differ  essentially  in  this  respect  from  the  saprophytic  types. 

It  is  necessary  to  consider  briefly  the  method  of  the  interchange 
of  material  between  the  vegetable  and  animal  kingdoms  in  order  to 
understand  the  full  significance  of  bacterial  action  in  the  economy  of 
nature.  All  animals  require  preformed  organic  compounds  for  their 
sustenance.  They  are  unable  to  build  up  these  compounds  of  which 
their  tissues  are  composed  from  chemical  elements  or  from  simple 
inorganic  salts.  They  are,  therefore,  dependent  directly  or  indirectly 
upon  the  synthetic  activities  of  green  plants  for  their  foodstuffs.  The 
green  plants  by  virtue  of  the  chlorophyll  contained  within  their  leaves 


GENERAL  CHEMISTRY  OF  BACTERIA  57 

and  stems  possess  the  power  of  combining  CO2,  water  and  nitrogenous 
salts  under  the  influence  of  sunlight  directly  into  the  highly  complex 
proteins  and  carbohydrates  essential  for  animal  food.  These  products 
of  the  synthetic  activity  of  the  plants  are  utilized  by  the  animal 
kingdom  for  food;  directly  by  the  herbivora,  indirectly  by  the  carni- 
vora.  These  substances  are  either  broken  down  within  the  digestive 
tract  of  the  animal  body  and  reconstructed  to  form  the  tissues  and 
supply  energy  to  the  animal,  or  eliminated  as  excreta.  The  excreta 
of  animals  are  not  sufficiently  simple  in  composition,  as  a  rule,  to  be 
used  directly  by  plants,  and  the  tissues  of  dead  animals  and  plants 
are  of  little  value  in  their  complex  state  for  plant  foods.  Further 
cleavage,  both  of  the  excreta  of  animals  and  the  dead  bodies  of  plants 
and  animals,  is  necessary  to  make  the  elements  contained  within  them 
utilizable  by  plants,  and  this  cleavage  is  brought  about  by  bacterial 
activity.  Various  saprophytic  bacteria  act  successively  upon  these 
complex  organic  compounds,  changing  them,  chiefly  by  hydrolytic 
cleavage,  into  stable,  fully  mineralized  salts,  which  are  directly  utiliz- 
able in  this  state  by  the  chlorophyll-bearing  plants.  There  is,  there- 
fore, a  constant  rotation  of  the  various  elements  which  enter  into  the 
composition  of  animal  and  plant  tissues  between  the  plant  and  animal 
kingdoms  respectively  by  means  of  an  anabolic  or  constructive  process 
in  the  one  (plants),  and  a  catabolic  or  destructive  process  in  the  other 
(animals).  The  cycle  as  outlined,  however,  is  not  a  continuous  one, 
for  there  are  important  gaps  in  the  process  of  cleavage  and  in  the  pro- 
cess of  synthesis  which  if  left  unbridged  by  the  bacteria  would  eventu- 
ally arrest  all  vital  activity  both  of  plants  and  animals,  and  all  life 
would  then  inevitably  cease  on  this  planet.  These  gaps  between  the 
animal  and  vegetable  kingdoms  are  filled  by  the  analytical  activity 
of  bacteria. 

A  small  group  of  bacteria,  on  the  other  hand,  is  also  important 
from  the  synthetical  point  of  view.  A  certain  amount  of  nitrogen  is 
lost  in  the  animal  and  vegetable  kingdoms  by  various  natural  agencies, 
and  this  supply  of  nitrogen  must  be  made  good  from  sources  which 
are  not  directly  available  either  to  plants  or  to  animals.  Approxi- 
mately 80  per  cent,  of  the  atmosphere  is  made  up  of  nitrogen,  and  a 
certain  group  of  bacteria,  "the  nitrogen-fixation"  bacteria  so-called, 
which  are  found  chiefly  on  the  nodules  or  roots  of  leguminous  plants, 
are  able  to  draw  upon  this  great  reservoir  of  atmospheric  nitrogen 
and  synthesize  it  into  nitrogen-containing  compounds  which  plants 
can  utilize  directly. 


58  THE  CHEMISTRY  OF  BACTERIA 

Another  type  of  bacterial  activity  of  importance  is  the  oxidation 
of  ammonia,  the  final  step  in  the  degradation  of  protein,  into  nitrites 
and  nitrates.  This  is  carried  on  by  the  nitrifying  bacteria  of  the  soil. 
Contrary  to  the  generally  accepted  idea,  therefore,  the  activities  of 
the  majority  of  bacteria  are  not  in  opposition  to  the  activities  of  man, 
animals,  and  plants;  bacteria  are  indispensable  agents  in  the  economy 
of  nature. 

B.    CHEMICAL   COMPOSITION   OF   BACTERIA. 

1.  Elementary  Composition. — Bacteria  normally  contain  the  same 
elements  in  their  substance  that  the  higher  plants  and  animals  contain, 
viz.,  carbon,  nitrogen,  hydrogen,  oxygen  and  phosphorus,  together 
with  smaller  amounts  of  sodium,  chlorine,  sulphur,  potassium,  calcium, 
magnesium,  and  traces  of  iron. 

2.  Chemical  Constitution. — The  elements  carbon,  hydrogen,  nitro- 
gen and  oxygen,  and  to  a  certain  extent  phosphorus,  and  perhaps 
sulphur  are  united  to  form  proteins,  nucleoproteins,  carbohydrates, 
and  fats.    The  inorganic  substance  of  bacteria  is  made  up  of  the  other 
elements  mentioned  above  in  variable  proportions.    Of  these  elements, 
carbon,  hydrogen,  nitrogen,  oxygen  and  phosphorus  are  the  most 
important.1 

TABLES   ILLUSTRATING   THE   CHEMICAL   COMPOSITION    OF 
BACTERIA. 

1.  .PERCENTAGE  OF  THE  ELEMENTS  IN  ASH-FREE  "  MYCOPROTEiN."2 

C  H  N 

per  cent.  per  cent.  per  cent. 

52.1-52.6  7.3-7.38  14.5-14.9 

2.  PERCENTAGE  COMPOSITION  WITH  RESPECT  TO  ORGANIC  AND  INORGANIC 
CONSTITUENTS. 

Putrefactive  Bacillus  Tubercle 

bacteria.3  prodigiosus.4  bacilli.5 

Water 83.42  85.45  85.00 

Protein 13.96  10.33  8.50 

Extractive 1.00  0.70  4.00 

Ash 0.78  1.75  1.40 

Residue 0.84  1.77  1.10 

1  Certain  acid-fast  bacteria  can  be  grown  in  media  containing  theoretically  but  five 
elements:   carbon,  hydrogen,  nitrogen,  oxygen,  and  phosphorus.     Lowenstein,  Centralbl. 
f.  BakterioL,  Original,  1913,  Ixviii,  591.      Wherry,  Centralbl.  f.  Bakteriol.,  1913,  Ixx, 
115.     Kendall,  Day  and  Walker,  Jour.  Inf.  Dis.,  1914,  xv,  428. 

2  Kruse,  Allgemein.  Microbiol.,  p.  62. 

3  Nencki  and  Scheffer,  Ueber  die  chemische  Zusammensetzung  der  Faulnisbakterien, 
Beitr.  z.  Biol.  d.  Spaltpilze.     Nencki,  Leipzig,  1880,  Jour.  f.  prakt.  Chemie,  N.  F.,  xix, 
u.  xx. 

4  Kappes,  Analyze  d.  Massen  Kulturen  einiger  Spaltpilze  u.  d.  Soorhefe,  Leipzig,  Diss., 
1889. 

5  Ruppel,  Die  Proteine,  1900,  Heft  4,  Beitr.  z.  exp.  Therapie.,   Ztschr.  f.  physiol. 
Chemie,  xxvi. 


CHEMICAL  COMPOSITION  OF  BACTERIA  59 

COMPOSITION   OF  BACTERIA.1 


Water. 

In  per  cent,  dry  residue. 
Acetone           CHCla 

Phosphorus, 
per  cent. 

per  cent. 

N 

extract. 

extract.3 

in  fat.4 

Glanders 

.      .      .      76.5 

10.5 

11.7 

8.6 

2.5 

Chicken  cholera     . 

.      .      .     79.3 

10.8 

7.5 

6.3 

2.4 

Cholera 

73  4 

9.8 

8.7 

6.8 

2.4 

Dysentery  (Shiga) 

.      .      .     78.2 

8.9 

12.8 

10.6 

1.6 

Proteus  vulgaris 

.      .      .      80.0 

10.7 

10.9 

7.1 

1.6 

Typhoid      .      .      . 

.      .      .      78.9 

8.3 

15.4 

10.6 

1.2 

Anthrax2 

.      .      ,     81.7 

9.2 

6.3 

1.5 

0.9 

Pseudotuberculosis 

.      .      .     78.8 

10.4 

15.6 

10.3 

0.8 

B.  pneumonias 

.      .      .      85.5 

10.4 

15.4 

10.3 

0.8 

B.  coli    .... 

.      .      .     73.3 

8.3 

15.2 

11.8 

0.8 

B.  prodigiosus 

.      .      .      78.0 

10.5 

9.0 

6.6 

0.5 

B.  psittacosis    . 

.      .      .     78.0 

9.5 

11.1 

7.0 

0.5 

B.  diphtherias 

.      .      .      84.5 

7.0 

5.2 

0.2 

B.  pyocyaneus 

.      .      .     75.0 

9.8 

15.8 

10.7 

0.2 

It  will  be  seen  that  from  75  to  86  per  cent,  of  the  bacterial  celt  is 
water.  The  remainder  of  the  cell  consists  chiefly  of  protein,  carbo- 
hydrate-like bodies,  extractives  (fats,  fatty  acids,  waxes  and  lipoids), 
and  inorganic  salts.  Of  these,  the  nitrogenous  substances  vary  greatly 
in  amount,  depending  upon  the  composition  of  the  medium  in  which 
the  organisms  are  grown.  _  The  extractives  (fats,  waxes,  lipoids,  and 
fatty  acids)  are  most  prominent  in  the  tubercle  bacillus  and  the  acid- 
fast  group.  Some  extractives,  however,  are  found  in  all  bacteria, 
they  being  greater  in  amount  on  a  medium  containing  carbohydrate 
and  protein  than  on  one  containing  protein  alone.  The  chemical 
determination  of  the  extractives  is  very  unsatisfactory,  partly  because 
of  the  difficulty  in  breaking  up  the  cell  sufficiently  to  facilitate  the 
entrance  of  the  solvent. 

3.  Chemical  Composition  of  Bacteria. — The  percentages  of  the  ele- 
ments and  various  constituents  of  bacteria,  as  indicated  in  the  above 
tables,  is  at  best  only  approximate.  Other  factors  very  markedly 
influence  the  composition  of  the  organisms. 

Of  these,  the  age  of  the  culture,  the  temperature  at  which  it  is 
grown,  and  the  composition  of  the  medium  in  which  the  organisms 
are  grown  are  the  most  important.  Generally  speaking,  young  cul- 
tures appear  to  contain  rather  more  dry  residue  than  older  cultures, 
and  bacteria  grown  at  37°  C.  contain  more  dry  residue  than  those 
grown  at  20°  C.5  The  inorganic  constituents  of  the  broth  influence 

1  Nicolle  and  Alilaire,  Ann.  1'Inst.  Past.,  1909,  xxiii,  547. 

2  Asporeless.  3  From  acetone  extract.  <  From  CHCls  extract. 

5  The  decrease  in  dry  residue  observed  in  old  cultures  is  partly  attributable  to  auto- 
lysis  of  bacteria ;  this  is  usually  observed  earlier  in  cultures  maintained  at  37  °  C.  than  in 
corresponding  cultures  kept  at  20°  C.  Growth  is  more  rapid  at  this  higher  temperature, 
and  recessive  changes  due  partly  to  the  accumulation  of  waste  products  are  seen  earlier. 


60 


THE  CHEMISTRY  OF  BACTERIA 


the  composition  of  bacteria  markedly.  Cramer1  has  found  that  the 
percentage  composition  of  the  ash  of  the  cholera  vibrio  varies  within 
very  considerable  limits  as  the  organism  is  grown  under  different 
conditions.  The  following  table  indicates  in  a  general  way  the  influ- 
ence of  these  factors: 


IS 


=4 
II- 


9.30 

22.30 

25.90 

1.34 

2.75 

3.73 

1.25 

2.50 

4.12 

28.70 

34.80 

10.90 

7.90 
16.90 

39.80 
7.97 

2.10 
46.70 

23.00 

11.40 

49.20 

Ash  content  of  bacteria  in  dry  substance 
Ash  content  of  moist  mass  .... 
Ash  content  of  medium  in  moist  mass 
Phosphoric  acid  in  bacterial  ash     . 
Phosphoric  acid  in  media  ash 
Chlorine  in  bacterial  ash      .... 
Chlorine  in  media  ash 


The  phosphorus  content  of  the  medium  in  these  experiments,  as 
shown  in  the  above  table,  was  varied  almost  twenty  times,  but  in  the 
bacterial  organisms  it  varied  scarcely  three  times.  The  variation  in 
chlorine  content  was  somewhat  greater. 

Even  as  important  an  element  as  nitrogen  is  subject  to  rather  wide 
variations  in  bacteria,  as  Cramer2  and  Lyons3  have  shown.  The  fol- 
lowing tables  summarize  Cramer's  and  Lyons's  results.  They  were 
obtained  by  growing  certain  bacteria  mentioned  specifically  below  on 
a  medium  consisting  fundamentally  of  1.5  per  cent,  agar,  to  which  were 
added  various  substances,  as  indicated  in  the  tables,  respectively 
Media  A,  B,  and  C.  The  general  procedure  was  to  grow  the  bacteria 
at  37°  C.  for  several  days,  to  wash  them  off  with  salt  solution,  to  free 
them  from  adherent  media  by  centrifugalization  and  washing,  to  dry 
the  washed  organisms  in  vacua  to  constant  weight,  and  to  analyze 
the  dry  residue  for  extractives  and  ash. 

CRAMER. 


Nitrogen  substance. 

Ether-alcohol 
extractives. 

Ash. 

•»*•         !• 

A 

B 

C 

A 

B 

C 

A 

B 

C 

Organism. 

Pfeiff  er  bacillus      .      . 

66.6 

70.0 

53.7 

17.7 

14.63 

24.0 

12.56 

9.10 

9.13 

Bacillus  H-284       .      . 

73.1 

79.6 

59.0 

16.9 

17.83 

18.4 

11.42 

7.79 

9.20 

Pneumonia  bacillus     . 

71.7 

79.8 

63.6 

10.3 

11.40 

22:7 

13.94 

10.36 

7.88 

Rhinoscleroma  bacillus 

68.4 

76.2 

62.1 

11.1 

9.06 

20.0 

13.45 

9.33 

9.44 

1  Quoted  by  Kruse,  Allgemeine  Mikrobiologie,  p.  88. 

2  Arch.  f.  Hyg.,  1893,  151.  3  Ibid.,  1897,  xxiii,  30. 


4  From  water. 


COMPOSITION  OF   THE  BACTERIAL  CELL 


61 


LYONS. 


Medium. 

Nitrogen- 
containing 
substance. 

Ether 
extractives. 

Alcohol 
extractives. 

Ash. 

A 

B 

C 

A 

B 

C 

A 

B 

C 

A 

B 

C 

Organism. 

Pfeiffer  bacillus    . 

62.75 

58.88 

45.88 

1.68 

3.502.67 

12.17 

17.30 

29.60 

7.16 

2.79 

3.09 

Bacillus  No.  281  .      . 

71.8159.12 

46.25 

3.32 

3.842.84 

11.39 

15.19 

22.78 

6.51 

3.66 

4.18 

"Thread  bacillus."    . 

61.06 

44.31 

33.25 

1.74 

2.24 

1.87 

18.40 

21.80 

27.50 

8.09 

4.50 

3.02 

Medium  A  agar,  1.5  per  cent. 
Medium  B  agar,  1.5  per  cent. 
Medium  C  agar,  1.5  per  cent. 


peptone,  1  per  cent, 
peptone,  5  per  cent, 
peptone  1  per  cent.;  dextrose,  5  per  cent. 


It  will  be  seen  that  the  nitrogen  content  of  the  bacteria  grown  in 
a  medium  containing  nitrogen  plus  carbohydrate  is  almost  25  per  cent, 
less  than  the  nitrogen  content  in  the  same  organisms  grown  in  the  same 
nitrogen  medium  but  with  no  carbohydrate.  The  nitrogen  content  is 
greatest  in  the  carbohydrate-free  medium,  the  extractives  are  greater 
in  the  carbohydrate-containing  medium.  This  decrease  in  the  nitrogen 
content  in  pathological  bacteria  grown  in  sugar  media  may  be  of 
considerable  importance,  particularly  in  the  preparation  of  vaccines 
and  other  antigens.  Nothing  is  known  definitely  of  the  distribution 
of  nitrogen  in  bacteria,  but  this  reduction  of  25  per  cent,  in  the 
nitrogen  content  may  well  influence  somewhat  the  immunizing  value 
of  vaccines. 


C.    COMPOSITION  OF  THE  MORPHOLOGICAL   COMPONENTS  OF 
THE   BACTERIAL   CELL. 

1.  Cell  Membrane. — Typical  cells  of  higher  plants  contain  cellulose, 
and  bacteria  were  formerly  differentiated  sharply  from  the  plant 
kingdom  because  cellulose  could  not  be  found  in  them.  Later  observa- 
tions would  suggest  that  cellulose  or  substances  chemically  closely 
related  to  it  are  demonstrable  in  certain  bacteria.  Dreyfuss2  appears 
to  have  identified  cellulose  in  bacteria  from  pus  and  in  B.  subtilis; 
Hammerschlag3  claims  to  have  isolated  cellulose  from  tubercle  bacilli, 
Dzierzgowski  and  Rekowski4  appear  to  have  found  cellulose  in  diph- 
theria bacilli;  more  recently  Tamura5  has  demonstrated  a  hemi-cellulose 

1  From  water. 

2  Ztschr.  f.  phys.  Chemie,  1893,  xviii,  375. 

3  Sitzber.  Akad.  Wiss.,  Wien,  xiii,  12. 

4  Arch.  Soc.  Biol.,  St.  Petersburg,  1892. 

6  Ztschr.  f.  phys.  Chem.,  1914,  Ixxxix,  289. 


62  THE  CHEMISTRY  OF  BACTERIA 

in  the  same  organism.  So  that  the  ability  of  at  least  certain  bacteria 
to  elaborate  cellulose  can  hardly  be  doubted. 

Emmerling1  identified  chitin  in  Bacterium  xylinum,  and  Irvanoff2 
gives  the  following  percentage  composition  of  the  cell  membranes  of 
B.  pyocyaneus,  B.  megatherium  and  B.  anthracis:  C,  46  per  cent.; 
H,  6.7-7  per  cent. ;  N,  8.4-8.8  per  cent. ;  which  is  empirically  very  similar 
to  chitin.  Chitin  is  chemically  a  polymer  of  glucoseamine,  CH2OH.- 
(CHOH)3.CHNH2.CHO,  which  in  turn  is  an  amino  hexose  very  similar 
to  dextrose,  except  that  it  has  an  amino  group  adjacent  to  the  aldehyde 
gro'up.  Chitins  are  typically  animal  in  origin,  and  are  rarely,  if  ever, 
found  in  typical  plants,  hence  the  distribution  between  cellulose  and 
chitin  in  bacteria  is  important  as  suggesting  relationships  to  the 
vegetable  or  animal  kingdoms. 

Many  bacteria  stain  brown  with  iodin,  and  the  assumption  is  that 
the  cell  membrane  of  such  organisms,  or  the  cell  substance  contains 
substances  similar  to  glycogen.  According  to  Arthur  Meyer,3  many 
bacteria  color  blue  with  very  small  amounts  of  iodin;  brown  or 
red-brown  with  an  excess  of  iodin;  indicating  that  there  is  a  very 
small  amount  of  starch  and  a  relatively  large  amount  of  glycogen 
or  amylodextrin  in  the  substance.  Similar  observations  have  been 
made  by  Heinze4  and  Levene,5  who  have  isolated  a  substance  from 
tubercle  bacilli  which  reacts  chemically  like  glycogen. 

2.  Capsule. — The  capsules  of  the  capsule-forming  bacteria  contain 
considerable  amounts  of  a  mucinous  substance  apparently  a  glyco- 
protein.    Cultures  of  bacteria  which  do  not  ordinarily  exhibit  capsules 
occasionally  produce  spontaneously  viscid,  mucinous  substances  in 
artificial  media;  thus,  strains  of  rabbit  septicemia  bacilli  and  glanders 
bacilli  may  become  viscid  after  repeated  transfers.6    Broth  cultures 
of  tubercle  bacilli  may  similarly  become  mucinous.7   Rettger's  observa- 
tions8 make  it  very  probable  that  these  viscid  substances  are  true 
mucins. 

3.  Cytoplasm. — The  cytoplasm  of  bacteria  consists  chiefly  of  the 
bacterial  protein,  which  appears  to  be  specific  in  character  for  any 

1  Berichte  d.  chem.  Gesell.,  1899,  541. 

2  Hofmeister's  Beitrage,  1902,  i,  524. 

3  Flora,  1899. 

4  Centralbl.  f.  Bakteriol.,  2te  Abt.,  1903,  xii;    1904,  xiv. 
6  Jour.  Med.  Research,  1901,  vi,  135. 

6  Theobald  Smith,  Transactions  of  First  Annual  Meeting  of  National   Association 
for  the  Study  and  Prevention  of  Tuberculosis. 

7  Weleminsky,  Berl.  klin.  Wchnschr.,  1912,  xlix,  1320;    Kendall,  Walker  and   Day, 
Jour.  Infec.  Dis.,  1914,  No.  11. 

8  Jour.  Med.  Research,  1903,  x,  101. 


COMPOSITION  OF  THE  BACTERIAL  CELL  63 

given  organism,  together  with  enzymes  and  at  least  minimal  quantities 
of  all  the  products  of  its  metabolism. 

Regarding  the  nature  of  the  protein  substance  in  bacteria,  but  little 
is  known,  although  50-80  per  cent,  of  the  dried  substance  of  the 
bacterial  cell  consists  of  protein  and  protein  derivatives.  Conspicuous 
among  these  protein  derivatives  are  the  nuclein  constituents,  nucleins, 
nucleoproteins,  and  nucleic  acids;  they  occur  constantly  in  bacteria 
and  apparently  the  greater  part  of  the  protein  of  the  bacterial  cell 
consists  of  these  nuclear  constituents.  Nucleins  and  nucleoproteins 
have  been  isolated  from  many  bacteria:  from  B.  subtilis  by  Van  de 
Velde;1  from  the  plague  bacillus  by  Lustig  and  Galeotti;2  from  the 
typhoid  bacillus  by  Paladino-Blandini;3  from  the  tubercle  bacillus 
by  Von  Ruck4  and  Ruppel;5  from  the  diphtheria  bacillus  by  Aronson;6 
and  Carapelle7  has  identified  a  glyco-nucleo-protein  in  B.  prodigiosus. 

Numerous  observations  indicate  that  nuclein  bases  (xanthin  bases) 
are  found  in  bacterial  cells;  thus,  Lustig  and  Galeotti8  identified 
xanthin  in  plague  bacilli.  Nashimura9  obtained  xanthin  bases  in  the 
dried  residue  of  a  water  bacillus  in  the  following  amounts:  xanthin  0.07 
per  cent.;  guanin,  0.14  per  cent.;  adenin,  0.08  per  cent.  No  hypox- 
anthin  was  found. 

The  amino-acids  of  bacterial  protein  have  not  been  thoroughly 
studied.  The  variable  nitrogen  content  even  of  the  same  organism 
as  it  is  grown  in  different  media  and  under  different  conditions  would 
suggest  that  quantitative  determinations  of  nitrogenous  substances 
would  be  somewhat  unsatisfactory.  Qualitatively,  so  far  as  available 
data  show,  many  amino-acids  found  in  protein  of  higher  animals  and 
plants  have  been  isolated  or  identified  in  bacterial  cells.  These  amino- 
acids  appear  to  differ  in  amount  in  different  organisms,  and  several 
have  not  been  isolated  at  all  up  to  the  present  time.  Vaughan,  Wheeler, 
and  Leach10  conclude  that  the  bacterial  substance  contains  carbo- 
hydrates, nuclein  bodies  and  polymers  of  mono-  and  diamino-acids. 
They  are  glyco-nucleo-proteins.  Kruse11  and  Vaughan12  have  arrived  at 

1  Ztschr.  f.  phys.  Chem.,  viii. 

2  Deutsch.  med.  Wchnschr.,  1897,  225. 

5  Baumgarten's  Jahresberichte,  1901,  228,  ref. 

4  Prophylactic  Immunization  against  Tuberculosis,  Report  No.  1,  Asheville",  1912,  3. 

6  Ztschr.  f.  phys.  Chem.,  1898,  xxvi. 

6  Arch.  f.  Kinderheilkunde,  vol.  xxx. 

7  Centralbl.  f.  Bakteriol.,  1907,  xliv,  440. 

8  Loc.  cit. 

9  Arch.  f.  Hyg.,  xviii,  325. 

10  Tr.  Assn.  Am.  Phys.,  1902,  p.  243. 

11  Allgemeine  Microbiologie,  p.  65. 

12  Protein  Split  Products,  p.  437. 


64  THE  CHEMISTRY  OF  BACTERIA 

the  same  conclusion.  The  analysis  of  one  hundred  grams  of  dried 
tubercle  bacilli  by  Ruppel1  indicates  the  importance  of  the  nucleins  in 
bacterial  proteins. 

Grams. 

Nucleic  acid  (tuberculinic  acid) 8.5 

Nucleoprotamin 25.5 

Nucleoproteid 23.0 

Albuminoids  (keratin,  etc.) 8.3 

Fat  and  wax 26 . 5 

Ash 9.2 

Carbohydrates. — Glycogen  or  some  similar  carbohydrate,  which  is 
readily  detected  by  the  mahogany  color  it  gives  with  iodine,  is  found 
in  many  bacteria,  as  has  been  stated  previously,  but  it  is  extremely 
difficult  to  decide  definitely  whether  it  is  limited  exclusively  to  the  cell 
membrane  or  scattered  somewhat  diffusely  through  the  cytoplasm 
as  well. 

Fats  and  Fatty  Derivatives. — Fats,  fatty  acids,  lipoids  and  waxes, 
which  may  be  demonstrated  by  staining  bacteria  with  Sudan  III, 
Scharlach  R,  and  osmic  acid,  occur  in  variable  amounts  in  the  tubercle 
bacillus  and  other  acid-fast  bacilli.  The  amount  of  these  extractives 
may  be  very  great  in  the  acid-fast  group,  varying  from  26  to  40  per  cent, 
of  the  total  dry  residue.  Considerable  discussion  has  centred  around 
the  distribution  of  these  substances,  many  authorities  claiming  that 
the  fats  and  waxes  are  contained  in  the  cell  wall  of  the  organism,  while 
others  maintain  that  these  substances  are  scattered  throughout  the 
cell  substance  as  well.  In  the  acid-fast  bacilli  it  is  probable  that  these 
fats  are  both  intra-  and  extracellular,  for  analyses  show  that  a  certain 
amount  of  them  can  be  extracted  from  intact  bacilli,  while  still  more 
can  be  extracted  when  the  organisms  are  broken  up.  The  following 
table  from  Kresling2  illustrates  the  distribution  of  the  fatty  substance 
of  the  tubercle  bacillus: 

I.     CONTENTS  OF  THE   DRIED   TUBERCLE   BACILLI   IN   THE 
PREPARATION   OF  TUBERCULIN. 

Per  cent. 

Moisture  (dried  at  100 °-l  10°  C.) 3.9375 

Moisture  (dried  in  desiccator) 3 . 08 

Ash 2.55 

Nitrogen - 8.575 

Nitrogen-containing  substances  (albumin)  reckoned  by  multiply- 
ing the  amount  of  N  by  the  factor  6.25  (the  N  of  lecithin  and 
other  substances  soluble  in  chloroform,  benzol,  ether,  and 

alcohol  were  not  reckoned) 53 . 59 

Fatty  substances  in  medium  after  the  first  four  determinations  38.95 
Other  N-free  substances,  reckoned  as  the  difference     .      .      .      .     0 . 9725 

1  Loc.  cit. 

2  Centralbl.  f.  Bakteriol.,  1901,  xxx,  909, 


FOOD  RELATIONSHIPS  OF  BACTERIA  65 

II.     FATTY   SUBSTANCE   OBTAINED   BY   EXTRACTION   WITH 

CHLOROFORM,  POSSESSES   THE   FOLLOWING 

CHARACTERISTICS : 

Melting  point 46°  C. 

Acid  number ' 23.08 

Reichert-Meissl  number 2.007 

Hehner  number 74 . 236 

Saponification  number 60.70 

Ether  number 36 . 62 

Iodine  number  (according  to  Hubl) 9 . 92 

III.     THE    FATTY   SUBSTANCE   OBTAINED   BY   EXTRACTION   WITH 
CHLOROFORM   CONTAINS: 

Per  cent. 

Free  fatty  acids 14.38 

Neutral  fats  and  esters  of  fatty  acids 77.25 

Alcohols  separated  from  the  fatty  acid  esters  (with  melting  point 

43.5-44°  C.)  39.10 

Lecithin 0.16 

Cholesterin Not  determined 

Substances  directly  soluble  in  water 0.73 

Substances  soluble  in  water  which  are  formed  by  the  complete 

saponification  of  the  fatty  substances 25 . 764 

Inorganic  Constituents. — The  most  conspicuous  inorganic  element 
found  in  the  ash  of  bacteria  is  phosphorus,  and  the  content  of  phos- 
phorus, recovered  as  phosphoric  acid,  frequently  reaches  as  high  as 
half  the  total  ash  weight.  It  is  probable  that  a  considerable  part  of 
this  phosphorus  is  combined  with  nucleic  acid  to  form  nucleo-protein. 

4.  Spores. — The  chemical  composition  of  spores  is  not  well  deter- 
mined, but  the  generally  accepted  theory  is  that  they  contain  relatively 
less  water  and  consequently  a  greater  proportion  of  proteins  and  ash. 
Reinke1  has  suggested  that  the  sporoplasm  is  an  anhydride  of  the 
cytoplasm  of  the  vegetative  cell.  Sporulation  implies  that  relatively 
considerable  amounts  of  water  must  be  taken  up  by  the  spore  sub- 
stance in  order  to  regain  the  proportion  of  this  substance  found  in  the 
parent  organism. 

D.  FOOD   RELATIONSHIPS   OF   BACTERIA. 

1.  General. — Food  is  any  substance  which  a  living  organism  may 
utilize,  either  by  making  it  a  part  of  its  living  material  or  as  a  source 
of  energy.  Food  which  is  suitable  for  utilization  by  any  organism  must 
contain  all  the  elements  necessary  for  the  building  up  and  maintenance 
of  that  organism.  Analyses  of  bacterial  cells,  which  have  been  given 
in  preceding  tables,  show  them  to  be  made  up  of  the  same  elements 
as  those  of  the  higher  plants  and  animals;  viz.,  carbon,  hydrogen, 
oxygen,  nitrogen,  and  phosphorus,  together  with  smaller  amounts 

1  Quoted  by  Kruse,  Allgem.  MikrobioL,  p.  57, 


66  THE  CHEMISTRY  OF  BACTERIA 

of  sodium,  potassium,  sulphur,  calcium,  and  magnesjum.  Foods  to 
be  fully  suitable  for  bacterial  needs,  therefore,  should  contain  these 
elements.  It  should  be  stated,  however,  that  the  food  requirements 
of  bacteria  vary  within  wide  limits,  but  the  above  statements  are 
generally  applicable. 

2.  Sources  of  Food. — (a)  Nitrogen. — The  nature  of  the  compounds 
in  which  nitrogen  must  be  presented  to  bacteria  as  food  varies  greatly 
among  the  different  groups.  The  nodule  bacteria  found  in  the  nodules 
on  the  roots  of  many  leguminous  plants  actually  utilize  atmospheric 
nitrogen:  nitrifying  bacteria  found  chiefly  in  the  soil  derive  their 
nitrogen  requirement  chiefly  from  mineral  salts  which  are  oxidized 
through  their  activities  to  nitrites  and  eventually  to  nitrates.  From 
this  very  simple  source  of  nitrogen  these  bacteria  are  able  to  synthesize 
the  complex  nitrogen-containing  proteins  of  their  bodies. 

The  majority  of  bacteria,  including  not  only  the  saprophytic  organ- 
isms but  most  of  those  pathogenic  for  man,  animals,  and  plants  as 
well,  thrive  in  media  in  which  nitrogen  is  presented  to  them  as  peptones, 
albumoses,  or  even  certain  amino-acids;  in  other  words,  upon  the  pro- 
ducts of  protein  digestion.  The  more  strictly  pathogenic  organisms, 
as  the  gonococcus,  may  require  nitrogen  in  the  form  of  highly  specific 
tissue  proteins.  Generally  speaking,  animal  protein  or  its  derivatives 
is  more  easily  utilized  by  bacteria  than  protein  of  vegetable  origin. 

(6)  Carbon. — The  simplest  carbon  compound  which  occurs  naturally, 
CO2,  cannot  be  used  by  bacteria,  except  certain  nitrifying  bacteria, 
as  a  source  of  energy,  for  it  is  already  fully  oxidized.  The  carbon 
of  proteins  and  their  derivatives,  of  carbohydrates,  and  of  fats,  on  the 
contrary,  is  readily  utilizable  by  most  bacteria.  As  a  rule,  hydro- 
carbons of  the  aliphatic  series  are  not  attacked  by  the  microorganisms, 
but  compounds  containing  oxygen  as  well  as  carbon  and  hydrogen  are 
better  adapted  for  microbial  food.  Organic  acids,  as  acetic  acid, 
aspartic,  tartaric,  and  many  oxy acids  are  utilizable  by  some  bacteria. 
The  simpler  alcohols  can  be  used,  but  by  very  few  bacteria.  The 
complex  alcohols,  like  glycerin  and  mannite,  on  the  other  hand,  are 
available  food  materials  for  many. 

The  best  nitrogen-free  food  compounds  for  microorganisms  are  the 
carbohydrates,  particularly  those  containing  six  and  twelve  carbon 
atoms,  the  hexoses  and  bioses  respectively.  Carbohydrates  containing 
four,  five,  or  any  number  of  carbon  atoms  not  a  multiple  of  three  are 
usually  not  readily  attacked  by  bacteria.  Starches  and  cellulose  are 
not  generally  utilizable,  although  certain  types  of  organisms,  notably 


FOOD  RELATIONSHIPS  OF  BACTERIA  67 

those  found  in  the  intestinal  tracts  of  herbivora,  appear  to  decompose 
them  very  readily. 

(c)  Hydrogen. — Hydrogen  is  readily  obtained  by  microorganisms 
from  organic  compounds  containing  available  carbon,  nitrogen,  and 
hydrogen,  but  not  apparently  from  water. 

(d)  Oxygen. — Oxygen  is  indispensable  to  the  life  of  all  living  organ- 
isms as  a  source  of  energy  and  for  structural  purposes.    A  few  bacteria, 
the  obligately  aerobic  bacteria,  can  live  only  in  the  presence  of  free 
oxygen;  another  small  group,  the  obligately  anaerobic  bacteria,  live 
either  in  the  absence  of  free  oxygen  or  at  best  in  the  presence  of  minimal 
amounts  of  it;  more  than  minimal  amounts  of  free  oxygen  act  as 
specific  poisons  to  them.    The  majority  of  bacteria  are  facultative  with 
respect  to  their  oxygen  requirements;  that  is,  they  can  either  live 
in  the  presence  of  free  oxygen  or  derive  their  oxygen  needs  from 
organic  compounds,  usually  the  carbohydrates  or  proteins. 

(e)  Inorganic  Salts. — Inorganic  salts  are  used  by  bacteria  almost 
wholly  for  structural  purposes.     The  requirement  for  mineral  com- 
pounds is  very  little,  for  these  substances  do  not  on  the  average  make 
up  more  than  7  to  10  per  cent,  of  the  solid  matter  of  the  bacterial  cell. 
The  essential  elements  and  the  percentage  of  them  found  in  the  ash 
of  certain  bacteria  have  been  referred  to  previously,  and  it  was  stated 
that  the  amount  of  inorganic  salts  found  in  the  bodies  of  the  bacteria 
bore  a  rather  direct  relationship  to  the  salt  concentration  of  the  media. 
Of  the  inorganic  elements,  phosphorus  is  the  most  important,  for  it 
makes  up  nearly  50  per  cent,  of  the  ash.     Phosphorous  in  contra- 
distinction to  any  other  inorganic  salt  is  absolutely  indispensable  to 
bacterial   growth.     It   is   combined   organically   in   nucleo-proteins, 
glyconucleo-proteins,  and  nucleic  acids,  which  form  the  greater  part 
of  the  protein  of  the  bacterial  cell. 


CHAPTER  IV. 


BACTERIAL  METABOLISM. 


I.  GENERAL. 

II.  THE  NATURE  OF  BACTERIAL  MET- 
ABOLISM. 

III.  NITROGEN  METABOLISM. 

IV.  CARBON  METABOLISM. 

V.  QUALITATIVE    CATABOLIC    REAC- 
TIONS OF  BACTERIA. 

A.  In  Media  Containing  Only 

Utilizable  Nitrogenous 
Substances. 

B.  In  Media  Containing  Both 

Utilizable  Nitrogenous 
Substances  and  Utilizable 
Carbohydrates . 


VI.  THE  QUALITATIVE  INFLUENCE  OF 
UTILIZABLE       CARBOHYDRATES 
UPON    THE    ELABORATION    OF 
PROTEOLYTIC  ENZYMES. 
VII.  QUANTITATIVE  MEASURE  OF  BAC- 
TERIAL METABOLISM. 
VIII.  THE  SIGNIFICANCE  OF  BACTERIAL 
METABOLISM. 

IX.  FERMENTATION    AND    PUTREFAC- 
TION. 


I.     GENERAL  BACTERIAL  METABOLISM. 

Two  distinct  phases  may  be  recognized  in  the  life-history  of  a 
bacterial  cell;  an  anabolic  or  constructive  phase,  during  which  the 
cell  becomes  morphologically  complete;  and  a  catabolic,  vegetative, 
or  fuel  phase,  in  which  the  mature  organism  reacts  chemically  upon 
its  environment  to  provide  the  energy  (fuel)  necessary  for  the  main- 
tenance of  the  cell.  Chronologically,  the  anabolic  phase  precedes  the 
catabolic  phase;  that  is  to  say,  the  bacterial  cell  must  be  morpho- 
logically complete  before  it  can  bring  about  its  characteristic  energy 
transformations;  practically  the  two  phases  overlap  somewhat. 

The  actual  amount  of  material  required  for  the  anabolic  phase  of  the 
bacterial  cell  is  very  small,  for  the  actual  weight  of  the  average 
bacterium  is  but  0.000,000,0016  of  a  milligram,  approximately  (see 
page  25).  The  structural  phase  is  practically  ended,  aside  from  the 
replacement  of  comparatively  slight  losses  of  substance  incidental 
to  the  elaboration  of  soluble  enzymes  or  to  additional  requirements 
for  the  formation  of  structural  elements,  such  as  capsules,  when  the 
organism  is  morphologically  complete.  The  waste  incidental  to  the 
utilization  of  material  for  purely  anabolic  needs  is  likewise  very  small 
in  amount,  and  the  total  environmental  change  attributable  to  the 
purely  constructive  phase  of  bacterial  metabolism  is  slight  andordin- 
arily  disregarded.1 

» Kendall,  Jour.  Med.  Res.,  1911,  N.  S.f  xx,  140. 


GENERAL  BACTERIAL  METABOLISM  69 

The  amount  of  material  required  for  the  catabolic  (vegetative  or 
fuel)  phase  of  the  bacterial  cell,  on  the  contrary,  is  relatively  large. 
The  energy  requirement  of  cellular  organisms  varies  rather  with  the 
area  of  their  surface  than  according  to  their  actual  volume;  conse- 
quently, very  minute  organisms,  as  bacteria,  in  which  the  surface  is 
relatively  very  great  in  comparison  with  their  size,  would  require  much 
more  material  for  energy  purposes  than  for  structural  purposes.  For 
example,  the  total  surface  area  of  a  million  average-sized  cocci  (each 
1  micron  in  diameter)  would  be  approximately  3.1416  sq.  mm.;  the 
weight  of  these  organisms,  assuming  the  specific  gravity  to  be  1.030 
(which  is  reasonably  accurate),  would  be  about  0.00054  mg.  The 
combined  surface  of  all  the  cocci  in  an  actively  growing  broth  culture 
of  such  organisms  would  be  very  considerable.  It  must  be  remembered, 
however,  that  these  figures  do  not  carry  any  specific  basis  for  the 
measurement  of  bacterial  activity  in  terms  of  chemical  or  physical 
phenomena;  they  merely  express  in  a  very  general  manner  the  physical 
basis  for  the  apparent  disproportion  observed  between  the  size  of 
bacteria  and  the  amount  of  change  they  induce  in  their  environment. 

The  energy  phase  commences  theoretically  when  the  cell  is  morpho- 
logically complete,  and  it  is  a  continuous  process  which  ends  only  with 
the  death  of  the  cell.  It  may  be  reduced  to  a  minimum  when  the  cell 
enters  upon  a  latent  state  of  existence,  as  in  spore  formation;  it  is 
greatest  when  the  organism  is  growing  in  a  favorable  medium  at  the 
optimum  temperature,  and  it  is  restricted  proportionately  when 
environmental  conditions  become  unfavorable. 

The  life-history  of  a  culture  in  which  innumerable  bacteria  are 
growing  can  not  be  sharply  divided  into  the  anabolic  and  catabolic 
phases.  During  the  first  few  hours  after  inoculation,  however,  the 
anabolic  aspect  predominates;  later  the  catabolic  aspect  predominates. 
Thus,  colon  bacilli  inoculated  into  dextrose  broth  fermentation  tubes 
do  not  produce  gas  in  visible  amounts  during  the  first  few  hours  of 
incubation,  although  the  medium  gradually  becomes  turbid,  due  to  the 
rapid  multiplication  of  bacteria.  Somewhat  later  gas  formation  is 
observed,  and  it  then  proceeds  with  considerable  rapidity.  The 
production  of  gas  is  indicative  of  a  period  of  great  vegetative  activity 
in  which  large  numbers  of  mature  colon  bacilli  utilize  the  dextrose  for 
their  energy  requirements.  Still  later  the  production  of  gas  ceases, 
the  activities  of  the  organisms  diminish,  and  the  culture  finally  dies 
out  as  waste  products  accumulate  in  sufficient  amounts. 

Those  bacteria  habitually  pathogenic  for  man  induce  less  striking 
physical  and  chemical  changes  in  their  environment,  as  a  rule,  than 


70  BACTERIAL  METABOLISM 

do  the  saprophytic  types,  as  Theobald  Smith1  showed  long  ago.  Thus, 
typhoid  bacilli  are  relatively  inert  culturally;  they  form  no  gas  in 
sugar  media,  no  indol,  and  do  not  liquefy  gelatin;  on  the  contrary, 
B.  coli  and  even  more  strikingly  B.  proteus  are  characterized  by  strik- 
ing cultural  changes;  B.  coli  produces  deep-seated  changes  in  protein, 
resulting  in  the  production  of  indol;  it  produces  gas  from  sugar  media, 
but  it  does  not  liquefy  gelatin.  B.  proteus  behaves  much  like  B.  coli 
in  sugar  media,  but  liquefies  gelatin  as  well.  These  marked  changes 
in  the  composition  of  the  medium,  namely,  the  production  of  indol 
from  protein,  the  production  of  gas  from  sugar,  and  the  liquefaction 
of  gelatin,  are  all  phenomena  associated  with  the  vegetative  or  fuel 
phase  of  bacteria. 

H.     THE  NATURE  OF  BACTERIAL  METABOLISM. 

Chemically  considered,  the  anabolic  phase  of  bacterial  activity  is 
one  characterized  by  the  synthesis  of  relatively  simple  substances, 
chiefly  nitrogen-containing,  into  the  complex  specific  bacterial  proto- 
plasm through  a  series  of  synthetic  reactions  among  which  reductions 
and  condensations  appear  to  be  the  more  prominent.  It  is  very 
probable  that  many  of  these  condensation  reactions  are  hydrogenic 
in  nature;  that  is,  two  simpler  molecules  are  united  into  one  molecule 
of  greater  complexity  through  the  removal  of  hydrogen  and  oxygen 
from  them  in  the  proportions  to  form  water. 

As  simple  illustrations:  the  formation  of  lactose  from  a  molecule 
each  of  dextrose  and  galactose, 


C6Hi2O6  +  C6Hi2O6  =  Ci2H22Oii  +  H2O 
Dextrose.       Galactose.          Lactose. 

the  formation  of  a  polypeptid,  glycyl-glycin,  from  two  molecules  of 
glycocoll,2 

NH2.CH2.COOH  +  H.NH.CH2.COOH  =  NH2.CH2.CO.NH.CH2.COOH  +  H2O 
Glycocoll.  Glycocoll.  Glycyl-glycin. 

and  the  formation  of  the  glyceride  of  a  fatty  acid  from  glycerin  and 
acetic  acid  may  be  cited, 

CH2.OH  +  HOOC.CHa  =  CH2.O.O.CH3 

CH.OH   +  HOOC.CHs  =  CH.O.O.CHs   +  3  H2O 
I  I 

CH2.OH  +  HOOC.CHs       CH2O.O.CH3 
Glycerin.          Acetic  acid.  Triacetin. 

1  Fermentation  Tube,  Wilder  Quarter  Century  Book,  1893,  p.  219.     (See  also  Kendall, 
Day  and  Walker,  Jour.  Am.  Chem.  Assn.,  1913,  xxxv,  1201-1249,  for  analytical  data.) 
«  Fischer,  Ber.  d.  deutsch.  chem.  Gesell.,  1906,  xxxix,  530. 


NITROGEN  METABOLISM  71 

The  catabolic  phase  is  essentially  analytic;  it  is  characterized 
chemically  by  a  series  of  reactions  in  which  the  cleavage  of  more 
complex  compounds  to  simpler  ones  with  their  simultaneous  or  sub- 
sequent oxidation,  involving  the  liberation  of  energy,  is  a  noteworthy 
feature.  The  catabolic  phase  is  chiefly  a  series  of  oxidations  of  carbon 
and  hydrogen.  (For  illustrative  catabolic  reactions  see  infra,  pp.  73,  76.) 

m.     NITROGEN  METABOLISM. 

Bacteria,  like  all  known  living  things,  contain  nitrogen  in  their 
substance,  and  nitrogen  in  some  form  is  absolutely  indispensable  for 
the  building  up  of  their  structure.  Nitrogen,  in  other  words,  is  an 
absolutely  essential  element  in  the  constructive  phase  of  the  bacterial 
cell.  The  form  in  which  nitrogen  must  be  presented  to  bacteria  in 
order  to  be  utilizable  by  them  varies  with  the  kind  of  organism.  The 
nitrogen-fixing  bacteria  found  on  the  roots  of  leguminous  plants  can 
utilize  the  nitrogen  of  the  atmosphere;  some  nitrifying  bacteria  can 
utilize  the  nitrogen  of  ammonium  salts.  (These  two  groups  of  organ- 
isms appear  to  be  the  only  ones  which  can  oxidize  nitrogen.)  Many 
bacteria  can  obtain  their  nitrogen  from  amino-acids.  The  majority1 
of  bacteria  pathogenic  for  man  and  the  higher  animals  are  somewhat 
more  exacting  in  this  respect  and  require  more  highly  organized 
nitrogen,  as  peptones  and  proteoses,  while  a  small  group  of  obligately 
human  pathogenic  bacteria,  as  the  gonococcus,  grows  only  in  media 
containing  nitrogen  as  it  exists  in  the  highly  specialized  protein  of 
human  origin,  at  least  during  their  first  growth  outside  the  human 
body  on  artificial  media. 

he  vegetative  phase  of  bacterial  metabolism  is  essentially  a  series 
of  oxidations  of  carbon  and  hydrogen;  nitrogen  can  not  be  oxidized  by 
the  great  majority  of  bacteria,  and  consequently  it  appears  to  yield 
little  or  no  energy  to  them.  When  nitrogen-containing  compounds 
as  amino-acids,  peptones,  albumoses,  or  proteins  are  utilized  for  the 
energy  requirements  of  these  organisms,  the  nitrogen  (amino  nitrogen) 
is  usually  eliminated  from  the  ^mino-acid  complex  incidental  to  the 
oxidation  of  the  carbon  and  hydrogen;  the  nitrogen  thus  eliminated 
appears  in  soluble  form  in  the  culture  medium  as  ammonia.  This 
process  is  true  deaminization.  .  Nitrates  and  even  nitrites  may  be 
sources  of  energy  to  many  bacteria,  usually,  however,  because  of  their 
valuable  oxygen  content.  To  summarize,  bacteria  must  have  available 
nitrogen  for  their  structural  needs,, but  nitrogen,  except  for  the  nitrogen- 
fixing  and  nitrifying  bacteria,  is  not  as  a  rule  a  source  of  energy  to 
them,  because  the  great  majority  of  bacteria  can  not  oxidize  it. 


72  BACTERIAL  METABOLISM 

IV.     CARBON  METABOLISM. 

Carbon  is  an  important  structural  element  for  bacteria,  and  it  is 
equally  indispensable  as  a  source  of  energy,  for  the  oxidation  of  carbon 
is  an  important  feature  of  the  catabolic  activity  of  the  majority  of 
microorganisms.  The  reduced  form  in  which  this  element  is  present 
in  amino-acids  and  other  protein  derivatives  appears  to  be  particularly 
adapted  for  structural  purposes;  for  fuel  purposes  it  is  less  available, 
possibly  because  of  the  necessity  of  introducing  free  oxygen  into  the 
carbon  complex  to  provide  the  requisite  energy  for  the  vegetative 
activities  of  bacteria,  as  well  as  the  additional  amount  of  work  required 
to  eliminate  the  nitrogen  of  the  amino-acid  molecule  fdeaminization). 
It  is  generally  stated  that  bacteria  with  relatively  few  exceptions 
fail  to  grow  with  their  customary  vigor  in  sugar-free  media  from  which 
free  (atmospheric)  oxygen  is  excluded;  the  relative  absence  of  available 
oxygen  in  such  compounds  would  explain  this  phenomenon,  in  part 
at  least. 

The  carbohydrate  molecule,  which  contains  no  nitrogen  and  in  which 
the  carbon  is  already  partially  oxidized,  can  be  utilized  for  fuel  purposes 
by  most  bacteria  with  less  expenditure  of  energy  for  its  preparation 
than  can  be  the  case  with  most  amino-acids,  peptones,  or  proteins; 
for  this  reason  it  is  very  probable  that  utilizable  carbohydrate  is 
act6d  upon  by  many  bacteria  in  preference  to  protein  carbon.  In 
this  sense  utilizable  carbohydrate  protects  or  shields  protein  or  protein 
derivatives  from  bacterial  attack  for  their  fuel  requirements;  it  does 
not  protect  protein  from  bacterial  breakdown  to  supply  their  structural 
requirements,  however. 

The  net  result  of  this  selective  protective  action  of  carbohydrates 
for  protein  is  important  because  the  amount  of  material  required  to 
provkL*  energy  for  the  bacterial  cell  far  exceeds  the  amount  of  material 
required  to  build  up  the  bacterial  cell.  The  chemical  transformations 
incidental  to  the  anabolic  phase  of  bacterial  metabolism  are  insignificant 
in  amount  and  ordinarily  not  noticeable;  on  the  contrary,  the  chemical 
transformations  associated  with  the  catabolic  phase  of  bacterial 
metabolism  are  relatively  very  considerable  in  amount;  and  the 
nature  and  extent  of  those  chemical  reactions  which  are  associated 
with  the  transformation  of  material  for  energy  are  important  not 
only  for  the  identification  of  bacteria,  they  collectively  comprise  the 
important  specific  function  of  bacteria. 


QUALITATIVE  CATABOLIC  REACTIONS  OF  BACTERIA         73 

V.     QUALITATIVE  CATABOLIC  REACTIONS  OF  BACTERIA. 

The  chemical  changes  observed  in  cultures  of  ordinary  bacteria  are 
chiefly  those  associated  with  the  breakdown  of  organic  substances  for 
energy — they  are  reactions  of  the  catabolic  phase  of  bacterial  meta- 
bolism. It  should  be  again  emphasized  that  the  energy  reactions — the 
catabolic  reactions — are  those  which  are  most  profoundly  influenced 
by  the  composition  of  the  nutritive  substrate  upon  which  the  organisms 
are  grown. 

A.  Reactions  of  Bacteria  in  Media  Containing  Only  Nitrogenous 
Substances  (Proteins  or  Protein  Derivatives)  Which  are  Utilized 
for  the  Energy  Requirements  of  Bacteria. — Proteins  are  composed 
of  amino-acids,  of  which  some  seventeen  are  recognized.  Bacteria 
which  decompose  protein  appear  to  act  upon  these  amino-acids  in  the 
last  analysis,  and  several  types  of  reaction  are  recognized  at  the  present 
time.  Each  kind  of  organism  utilizes  protein  or  protein  derivatives 
somewhat  differently  and  characteristically,  but  in  general  one  or  more 
of  the  following  types  of  reactions  are  involved  either  successively  or 
simultaneously  in  the  catabolism  of  these  substances.  The  reactions 
follow  i1 

1 .  R.CH2.CHNH2.COOH  +  H2  =  R.CH2.CH2.COQH  4-  NH*    Re- 
ductive deaminization  of  amino-acid  to  fatty  acid   with  the    same 
number  of  carbon  atoms. 

2.  R.CH2.CHNH2.COOH  +  H2O  =  R.CH2.CHOH.COOH  +  NH3v 
Hydrolytic  deaminization  of  amino-acid  to  oxy-acid  with  the  same 
number  of  carbon  atoms. 

3.  R.CH2.CHNH2.COOH  +  O2  =  R.CH2.CO.COOH  +  NH3.    Oxi- 
dative  deaminization  of  amino-acid  to  keto-acid  with  same  number  of 
carbon  atoms. 

4.  R.CH2.CHNH2.COOH-R.CH2.CH2.HN2  +  CO2.      Carooxylic 
decomposition  of  amino-acid  to  amine  with  one  less  carbon  atom. 

5.  R.CH2.CH2.COOH  -*  R.CH2.CH3.  +  CO2.     Carboxylic  decom- 
position of  fatty  acid. 

6.  R.CH2.CH2.COOH  +  3O  =  CH2.COOH  +  CO2  +  H2O.     Carbo- 
xylic decomposition  with  the  formation  of  a  fatty  acid  with  one  less 
carbon  atom. 

A  few  illustrations  will  indicate  the  nature  of  these  changes  in  amino- 
acids  with  the  production  of  certain  substances  of  clinical  interest : 
1.  Formation  of  indol  from  tryptophan.    Indol  is  a  substance  pro- 

*•  See  Kruse,  Allgem.  Mikrobiol.,  505-536,  for  literature. 


74 


BACTERIAL  METABOLISM 


duced  in  the  intestinal  tract  from  tryptophan  (an  amino-acid  found 
in  protein),  chiefly  by  B.  coli  and  B.  proteus.  The  reactions  through 
which  tryptophan  is  changed  to  indol  by  these  organisms  are  as 
follows.1 


\ CH2.CHNH2.COOH 

+  H2  = 

Ax 

NH 

Tryptophan. 


CH2.CH2.COOH 


V  NH 

•  (deaininization) 
Indol  propionic  acid. 


+  NHs     Indol  propionic  acid 
+  3O  = 


/\ 


\  _  CHz.COOH 

+  CO2 


CHs 


H2O-> 


NH 

Indol  acetic  acid. 


C02 


Skatol  +  3  O 


NH 


Skatol. 


CO2  +  H2O 


NH 


Indol. 


Indol  contains  little  or  no  energy  for  most  bacteria,  and  it  is  left 
as  such  in  the  culture  medium  or  the  intestinal  tract.  Indol  is  fre- 
quently absorbed  from  the  intestinal  tract,  but  it  has  little  or  no 
energy  for  the  human  body — it  is  oxidized  in  the  liver  to  indoxyl 


and  is  excreted  and  appears 
in  the  urine  as  indican 


o 
/ 

O  - S  -  ONa 

II 
Q 


NH 


NH 


B.  coli,  B.  proteus,  and  other  organisms  which  "form  indol"  utilize 
the  alanin  radical  of  the  tryptophan  molecule  (alpha  amino  propionic 
acid)  for  energy,  first  eliminating  the  nitrogen  (deaininization),  then 
oxidizing  the  carbon.  The  indol  radical  which  is  left  is  not  a  source 
of  energy;  it  can  not  be  oxidized  by  these  organisms,  consequently 
it' remains  as  such  in  culture  media  or  is  absorbed  from  the  intestinal 
tract. 


Nencki,  Sitzungsber.  Wien.  Akad.,  1898,  II  Abt.,  xcviii,  412. 


QUALITATIVE  CATABOLIC  REACTIONS  OF  BACTERIA         75 
2.  Production  of  phenolic  bodies  from  tyrosine. 


OH 


+  NH3 

Paraoxyphenyl  propionic  acid 

+  3  O  = 
CH2.CHNH2.COOH  CH2.CH2.COOH 

Tyrosine.  Paraoxyphenylpropionic  acid 

(deaminization) 

OH  OH  OH 


C02  +  H20  ->  +  C02  +  C02 


Paracresol 

CH2.COOH  CH3      +  3  O  = 

Paraoxyphenyl  Paracresol.  Phenol, 

acetic  acid. 

Phenol  is  not  oxidizable  by  bacteria,  hence  it  remains  as  such 
unchanged  in  the  culture  media.  Phenol  (or  cresol)  may  be  absorbed 
from  the  intestinal  tract,  but  it  appears  eventually  in  the  urine  as  an 
ethereal  sulphate,  precisely  as  indol  appears  in  the  urine  as  indican. 
Indol  and  phenolic  bodies  are  not  found  in  cultures  containing  utiliz- 
able  carbohydrate — the  bacteria  which  produce  indol  and  phenols 
from  tryptophane  and  tyrosine,  respectively,  can  obtain  their  requisite 
energy  far  more  directly  and  economically  from  the  sugar  than  from 
the  nitrogen-containing  amino  acid.  Doubtless  the  same  general 
principle  applies  to  the  formation  of  these  aromatic  substances  in  the 
intestinal  tract. 

3.  Formation  of  amines  from  amino-acids  by  bacterial  action. 

(a)  Cadaverin  from  lysine.1 

CH2.CH2.CH2.CH2.CH.COOH  CH2.CH2.CH2.CH2.CH2 

I  I  -  I  I-      +C02 

NH2  NH2  NH2  NH2 

Lysine.  Cadaverin. 

(6)  Putrescin  from  ornithin.2 

CH2.CH2.CH2.CH.COOH  CH2.CH2.CH2.CH2 

|  |  -»•'/•      •"•!  I      +00, 

NH2  NH2  NH2  NH2 

Ornithin.  Putrescin. 

1  Ladenburg,  Ztschr.  f.  phys.  Chem.,  1886,  xix,  780. 

2Ellinger,  Ztschr.  f.  phys.  Chem.,  1902,  xxix,  334;  Ber.  d.  deut.  chem.  Gesell.,  1889, 
xxxi,  3183;  ibid.,  1900,  xxxii,  3542. 


76  BACTERIAL  METABOLISM 

(c)  Betaimidazoleethylamine  from  histidine. 

H— C— NHV  H— C— NHV 

\r*t¥  \r|fi 

II  //^™-  II  //^*± 

C— N    *  =  C— N    *  +  CO2 

I  I 

CH2  CH2 

I  I 

CHNH2  CH2NH2 

I 
COOH 

Histidine.  Betaimidazoleethylamine. 

According  to  Vaughan,1  betaimidazoleethylamine  is  possibly  the 
active  poisonous  principle  of  the  protein  molecule.  Recent  investiga- 
tions would  suggest  that  its  liberation  in  the  intestinal  tract  as  the 
result  of  bacterial  decomposition  of  protein  there  and  its  absorption 
into  the  body  may  be  associated  with  symptoms  of  considerable 
severity.  The  substance  is  not  formed  as  a  product  of  bacterial 
metabolism  in  media  containing  utilizable  carbohydrates. 

B.  Reactions  of  Bacteria  in  Media  Containing  Both  Utilizable 
Nitrogenous  Substances  (Protein  and  Their  Derivatives)  and  Carbo- 
hydrates.— Carbohydrates  contain  no  nitrogen;  consequently  pure 
carbohydrate  solutions  are  not  complete  foods  for  bacteria,  they 
are  important  chiefly  as  sources  of  energy  to  them.  Generally  speak- 
ing, carbohydrates  containing  two,  four,  five,  seven  or  eight  carbon 
atoms  are  not  readily  fermentable  by  bacteria.  Those  containing 
six  carbon  atoms,  particularly  dextrose,  are  most  readily  utilizable, 
those  with  three  carbon  atoms,  generally  speaking,  somewhat  less 
so.  Bioses,  containing  twelve  carbon  atoms,  and  starches  appear  to 
be  hydrolyzed  to  sugars  containing  six  carbon  atoms  before  they  are 
finally  oxidized. 

The  final  utilization  of  sugars  for  energy  by  bacteria  varies  accord- 
ing to  the  type  of  organism;  the  following  qualitative  reactions  are 
illustrative  of  some  of  the  general  types  of  decomposition  usually  met 
with.  It  must  be  remembered  that  the  exact  quantitative  utilization 
of  carbohydrates  by  bacteria  and  the  nature  and  composition  of  many 
of  the  intermediary  products  formed  from  them  are  still  uncertain. 

1.  C12H22On  +  H2O  =  C6H12O6  +  C6H12O6. 

Lactose.  Dextrose.  Galactose. 

Hydrolytic  cleavage  of  a  biose  to  two  molecules  of  hexose  sugar. 

2.  C6Hi2O6  =  3CH3COOH.     Pure  acetic  acid  fermentation. 

3.  C6H12O6  =  2CH3CHOHCOOH.     Pure  lactic  acid  fermentation. 

1  Protein  Split  Products. 


QUALITATIVE  CATABOLIC  REACTIONS  OF  BACTERIA        77 

4.  C6H12O6  =  CH3.CH2.CH2.COOH  +  2C02  +  2H2.    Pure  butyric 
acid  fermentation. 

5.  C6Hi206  =  2CH3CH£)H  +  2C02.    Pure  alcoholic  fermentation. 

6.  2C6H12OG   +  H20   =   2CH3.CH#)H.COOH  +  CH3.COOH   + 
C2H5OH'  +  2CO2  +  2  +  2H2.    The  type  of  fermentation  produced 
by  B.  coli  in  dextrose  broth.1 

The  sugars  containing  six  carbon  atoms  appear  to  be  somewhat 
more  utilizable  than  their  corresponding  alcohols:  thus,  the  Shiga 
bacillus  (B.  dysenteriae)  can  not  ferment  mannite;  it  can,  however,, 
readily  ferment  dextrose.  This  would  suggest  that  the  aldehyde 
group — CHO — is  somewhat  more  readily  attacked  than  the  alcohol 
group — CH2OH — ,  for  mannite  has  no  aldehyde  group  and  dextrose 
has  an  aldehyde  group.  The  alcohols  in  general  appear  to  be  less 
readily  acted  upon  by  bacteria  than  are  the  corresponding  aldehydes  or 
even  organic  acids,  provided  the  latter  are  not  too  greatly  dissociable. 

The  products  of  fermentation  of  higher  alcohols,  as  mannite,  by 
bacteria  are  somewhat  different  from  those  of  the  corresponding  sugars 
(aldoses).  The  chief  points  of  difference,  according  to  our  present 
knowledge,  consist  principally  in  the  production  of  more  alcohol 
when  the  'higher  alcohols  are  utilized  than  when  the  corresponding 
aldoses  are  concerned.  This  has  been  worked  out  satisfactorily  for 
certain  bacteria,  notably  the  colon  and  the  typhoid  bacilli,  by  Harden.2 
It  is  not  definitely  known  for  many  other  organisms.  The  gas-forming 
bacteria,  as  a  rule,  produce  more  gas  and  more  alcohol  from  the  alcohols 
of  the  Ce  series  than  from  their  corresponding  aldoses.  This  gas 
formation  appears  to  result  from  the  decomposition  of  formic  acid 
by  the  activity  of  a  specific  enzyme,  formiase,  according  to  the  equa- 
tion HCOOH  =  CO2  +  H2O.3  Thus,  B.  coli  and  related  gas-forming 
bacteria,  according  to  this  theory,  produce  the  ferment,  formiase, 
while  B.  typhosus,  which  also  produces  formic  acid  from  the  decom- 
position of  dextrose,  does  not  possess  this  ferment  and  consequently, 
forms  no  gas  in  sugar  solutions.  Formic  acid  is,  therefore,  somewhat 
prominently  represented  among  the  decomposition  products  of  carbo- 
hydrates by  the  typhoid  bacillus,  while  formic  acid  is  either  not 
present  or  present  in  small  amounts  in  corresponding  cultures  of  colon 
bacilli.4 

The  qualitative  changes  produced  in  fats  and  lipoidal  substances 
by  bacteria  are  not  well  known. 

1  Kruse,  Allgemeine  Mikrobiologie,  p.  294. 

2  Jour.  Hygiene,  1905. 

3  Franzen  and  Stuppuhn,  Ztschr.  f.  physiol.  Chem.,  1912,  Ixxvii,  129. 
«  Clark,  Science,  November  7,  1913. 


78  BACTERIAL  METABOLISM 

VI.     QUALITATIVE  INFLUENCE  OF  UTILIZABLE  CARBOHYDRATES 
UPON  THE  ELABORATION  OF  PROTEOLYTIC  ENZYMES. 

Certain  bacteria,  as  for  example  B.  proteus,  characteristically  pro- 
duce proteolytic  enzymes  which  rapidly  dissolve  gelatin  by  hydrolytic 
cleavage.  These  enzymes  are  exo-enzymes;  that  is,  they  may  be 
obtained  sterile  and  free  from  bacteria  simply  by  passing  gelatin 
liquefied  by  their  action  through  sterile  unglazed  procelain  filters. 
Although  the  bacteria  which  elaborated  the  enzymes  are  removed  by 
this  filtration,  the  sterile  filtrate  still  contains  the  active  enzyme  which 
will  liquefy  considerable  amounts  of  gelatin.  The  function  of  these 
enzymes  is  to  prepare  the  gelatin  for  assimilation  by  the  proteus  bacil- 
lus: the  gelatin  is  broken  down  by  enzyme  action  to  gelatin  peptone 
or  even  to  polypeptids.  The  proteus  bacillus  does  not  produce  soluble 
gelatin-splitting  enzymes  in  gelatin  containing  utilizable  carbohy- 
drate, although  sugar-free  gelatin  contains  them  in  considerable 
amounts.  These  gelatinases,  however,  will  liquefy  sugar-gelatin  quite 
as  readily  as  sugar-free  gelatin,  indicating  that  the  enzyme  itself 
is  not  inactivated  by  the  sugar,  at  least  in  the  amount  usually 
employed,  1  per  cent.  The  same  phenomenon  is  observed  in  cul- 
tures of  the  cholera  vibrio  and  many  other  bacteria  which  liquefy 
sugar-free  gelatin.  Extensive  investigations  by  Auerbach,1  and  by 
Kendall,  Day  and  Walker2  have  shown  that  the  gelatinase,  which, 
as  has  been  noted,  is  produced  only  in  sugar-free  gelatin,  although 
it  liquefies  sterile  sugar  gelatin,  prepares  protein  for  utilization  by 
these  bacteria  for  purely  catabolic  purposes;  if  the  organisms  have 
access  to  utilizable  carbohydrate  the  enzyme  is  not  produced  by 
them,  because  they  utilize  the  sugar,  not  the  protein,  under  these 
conditions  as  the  source  of  their  energy.  These  observations  indicate 
how  fundamentally  the  metabolism  of  bacteria  is  influenced  by  the 
nature  and  composition  of  the  substrate  upon  which  they  are  grown. 

VH.     QUANTITATIVE  MEASURE  OF  BACTERIAL  METABOLISM. 

It  is  possible  to  measure  the  nitrogen  metabolism  of  bacteria  under 
varying  conditions  with  a  very  considerable  degree  of  accuracy  in 
spite  of  the  minute  amounts  of  products  involved.  Such  measure- 
ments are  not  only  indicative  of  the  nature  and  degree  of  the  decom- 

1  Arch.  f.  Hyg.,  1897,  xxxi,  311. 

2  Jour.  Am.  Chem.  Assn.,  1914,  xxxvi,  1962. 


QUANTITATIVE  MEASURE  OF  BACTERIAL  METABOLISM     79 

position  of  purely  nitrogenous  substances  by  bacteria;  they  furnish 
quantitative  evidence  of  the  extent  of  the  utilization  of  carbohydrates 
by  bacteria  in  preference  to  nitrogenous  substances  for  fuel  (catabolic) 
purposes;  that  is  to  say,  such  measurements  evaluate  the  nitrogen 
metabolism  of  bacteria  in  purely  protein  solutions,  and  their  nitrogen 
metabolism  in  media  containing  both  protein  and  utilizable  carbo- 
hydrate. 

Such  determinations  have  been  made  for  a  large  series  of  bacteria 
by  Kendall  and  Farmer,1  and  Kendall,  Day  and  Walker.2  The  gen- 
eral method  followed  is  to  measure  the  amount  of  ammonia  (deamin- 
ization)  which  appears  in  fluid  cultures  of  bacteria  under  various 
conditions  of  growth.  The  following  table  shows,  respectively,  the 
change  in  reaction  (to  neutral  red  as  an  indicator  in  terms  of  T  acid 
or  alkali  per  100  c.c.  media)  and  the  increase  in  ammonia  (milli- 
grams per  100  c.c.  media),  as  certain  bacteria  are  grown  for  ten  days 
in  plain  and  dextrose  broth  respectively.  The  broths  are  identical 
in  initial  composition  and  reaction,  except  that  the  "dextrose  broth" 
contains  in  addition  to  the  ingredients  of  the  "plain  broth"  1  per  cent, 
of  chemically  pure  dextrose.  All  other  conditions  are  exactly  parallel. 
The  results  are  averages  of  several  strains  of  the  same  organism  in 
various  lots  of  media.  It  will  be  seen  that  B.  alcaligenes,  for  example, 
which  ferments  no  sugars,  produces  an  alkaline  reaction  (indicated 
as  "  "  in  the  table)  both  in  plain  and  dextrose  broth:  the  amounts 
of  ammonia  in  both  media  are  nearly  the  same. 

All  the  organisms  which  ferment  dextrose  produce  less  ammonia 
in  the  dextrose  medium  than  in  the  corresponding  sugar-free  medium, 
although  the  numbers  of  living  bacteria  were  found  to  be  greater  in 
the  former  than  the  latter.  The  small  amount  of  ammonia  in  the 
dextrose  broth  appears  to  be  largely  the  nitrogenous  waste  incidental 
to  the  utilization  of  protein  for  structural  purposes:  the  relatively 
large  amount  of  ammonia  observed  in  the  corresponding  sugar-free 
broths  is  the  combined  "structural  waste"  and  the  " deaminization" 
incidental  to  the  utilization  of  protein  for  their  energy  requirement. 
The  progressively  pathogenic  bacteria,  as  the  diphtheria,  typhoid 
and  dysentery  bacilli,  produce  much  Ies3  ammonia  in  sugar-free 
media  than  do  the  same  organisms  in  various  lots  of  media.3  (Kendall, 
Day  and  Walker.) 

1  Jour.  Biol.  Chem.,  1912,  xii,  13,  215,  219,  465;   xiii,  63.     Methods  ^iven  here. 

2  Jour.  Am.  Chem.  Soc.,  1913,  xxxv,  1201-1249. 

3  Ibid. 


80  BACTERIAL  METABOLISM 

Sugar-free  broth.  Sugar  broth. 

Ten-day  observations.  Reaction.1  NH.s  Reaction.1  NH.s 

B.  alcaligenes -1.25  +3.50  -1.15  +5.30 

B.  dysenterise  (Shiga)        ....  -0.30  +4.20  +2.80  0.00 

B.  dysenteries  (Flexner)    ...  -0.25  +3.10  +2.45  0.00 

B.  typhosus -0.45  +5.40  +3.30  +0.60 

B.  diphtheria  ......  —0.50  +3.10  +2.80  +1.05 

B.  of  hemorrhagic  septicemia      .  —0.20  +4.70  +2.25  +0.35 

B.  paratyphosus  alpha  and  beta  —0.10  +7.50  +3.90  +1.20 

B.  icteroides -0.10  +4.20  +3.80  +2.10 

B.  of  hog  cholera  avirulent    .      .  -1.25  +16.45  +3.70  +1.05 

B.  of  hog  cholera  virulent      .      .  -0.75  +8.40  +2.65  +1.05 

B.  of  fowl  cholera        ....  -    .00  +13.65  +3.35  +0.70 

B.  of  Morgan -    .33  +29.50  +3.90  +29. 662 

B.  coli -    .00  +24.40  +4.90  +0.35 

B.  cloacae -    .20  +39.20  -0.30  +36. 402 

B.  proteus  .      ...,..-    .98  +58.40  +3.55  +1.40 

Sp.  cholerse -    .45  +62.80  +2.00  +0.70 

Sp.  of  Finkler  and  Prior  .      .      .  -1.00  +27.30  +1.50  +0.70 

Sp.  of  Metchnikoff      .      .      .      .  -4.30  +41.30  +2.70  +0.70 

B.  pyocyaneus -1.85  +30.30  -1.33  +41.50 

Streptococcus +0.70  +1.40  +5.00  +0.70 

Staphylococcus -0.75  +38.70  +3.75  +0.70 

Mic.  tetragenus +1.00  +2.10  +3.00  +0.70 

Mic.  melitensis —0.10  +6.30  +3.50  +0.70 

Vm.  SIGNIFICANCE  OF  BACTERIAL  METABOLISM,  WITH  SPECIAL 

REFERENCE  TO  THE  SPARING  ACTION  OF  UTILIZABLE 

CARBOHYDRATE  FOR  PROTEIN. 

Considerable  emphasis  has  been  placed  upon  the  sparing  action 
of  utilizable  carbohydrate  for  protein  in  the  preceding  pages.  It  now 
remains  to  summarize  the  salient  features  of  this  aspect  of  bacteriology 
and  to  indicate  briefly  by  means  of  a  few  illustrations  precisely  how 
a  comprehension  of  the  principles  underlying  bacterial  metabolism 
may  be  made  use  of  in  controlling,  or  at  least  influencing  the  action 
of  these  microorganisms  upon  their  environment.  The  examples 
selected  are  chosen  rather  with  a  view  of  indicating  the  extreme  range 
of  the  subject  than  for  completeness  along  any  limited  line  of  inves- 
tigation. 

].  The  Composition  of  Bacteria. — Experiments  quoted  previously 
(page  60)  show  very  clearly  that  the  percentage  of  composition  of  the 
bacterial  cell  varies  according  to  the  medium  in  which  it  is  grown. 
Particularly  striking  is  the  difference  in  nitrogen  content  when  the 
same  bacterium  is  grown  in  media  of  the  same  nitrogenous  composition 
and  reaction  with  and  without  the  addition  of  utilizable  carbohydrate. 

1  Neutral  red,  —  =  alkaline  reaction,  +  =  acid  reaction. 

2  These  organisms  can  utilize  1  per  cent,  of  dextrose  without  forming  enough  acid 
to  inhibit  their  growth;    after  the  dextrose  is  used  up  they  attack  the  protein  for  their 
fuel  needs — hence  the  ammonia  production  in  a  medium  containing  utilizable  sugar. 
During  the  initial  period  when  sugar  is  present,  the  ammonia  value  is  very  little,  and 
the  reaction  is  acid. 


THE  SIGNIFICANCE  OF  BACTERIAL  METABOLISM  81 

2.  The  Recognition  of  Bacteria. — The  recognition  of  many  kinds 
of  bacteria,  as  for  example  members  of  the  intestinal  group,  depends 
upon  the  reactions  these  organisms  induce  in  various  sugars.    Thus, 
B.  alcaligenes  ferments  no  sugars;  B.  dysenterise  ferments  dextrose 
with  the  production  of  acid;  B.  proteus  ferments  dextrose  and  sac- 
charose with  the  evolution  of  gas  and  the  production  of  acid;  B. 
coli  ferments  dextrose  and  lactose  with  the  evolution  of  gas  and  the 
production  of  acid;  B.  coli  coagulates  milk,  while  B.  proteus  charac- 
teristically peptonizes  it.     All  of  these  reactions  are  explained  per- 
fectly upon  the  theory  that  utilizable  carbohydrate  protects  protein 
from  bacterial  breakdown.     Thus,   B.   alcaligenes  does  not  utilize 
any  carbohydrate;  as  is  well  known,  it  is  carnivorous.    B.  dysenterise 
can  utilize  dextrose,  and  consequently  it  produces  acid  in  a  medium 
containing   both   protein   derivatives   and   this   sugar:    similarly,  B. 
proteus  and  B.  coli  ferment  dextrose  and  in  addition  a  specific  biose. 
B.  proteus,  however,  does  not  ferment  lactose,  hence  it  attacks  the 
protein  of  milk;  while  B.  coli,  which  does  ferment  lactose,  produces 
an  acid  coagulation  in  milk:  the  acid  resulting  from  the  fermenta- 
tion of  the  milk  sugar   (lactose)  protects  the  proteins  of   the  milk. 
In  each  instance  the  organisms  attack  the  utilizable  carbohydrate 
whenever  it  is  present,  in  preference  to  the  protein  for  their  energy 
requirements.    If  bacteria  did  -not  habitually  utilize  carbohydrate 
in  preference  to  protein  for  their  fuel  needs,  these  fermentation  reac1- 
tions  would  be  of  no  value  whatsoever  as  diagnostic  tests  for  these 
various  microorganisms. 

3.  Certain  bacteria,  notably  B.  proteus,  produce  active,  soluble 
(extracellular)    enzymes    when    grown    in    sugar-free    gelatin,    that 
bring  about  an  energetic  liquefaction  of  this  medium,  which  becomes 
alkaline  in  reaction.    If  the  organisms  are  grown  in  dextrose  gelatin 
no  liquefaction  takes  place;  the  bacilli  produce  CO2  and  H2  as  well 
as  acid  in  dextrose  gelatin,  using  the  sugar  in  preference  to  the  protein 
for  their  energy  needs.    The  liquefied  gelatin  containing  the  soluble 
gelatinase  may  be  sterilized  by  passage  through  a  Berkefeld  filter, 
thus  removing  all  bacteria.     The  filtrate  will  liquefy  sterile  plain  or 
sterile  dextrose  gelatin,  thus  proving  that  the  soluble  enzyme,  which 
prepares  gelatin  for  assimilation  by  proteus  bacilli   (and  which  is 
only  produced  in  a  carbohydrate-free  medium),  acts  specifically  on 
the  protein  irrespective  of  other  substances  which  may  be  present. 
In  this  instance  the  presence  of  utilizable  sugar  in  cultures  of  living 
proteus  bacilli   protects  the  protein  (gelatin  in  the  instance  cited) 


82 


BACTERIAL  METABOLISM 


from  bacterial  attack,  and  inasmuch  as  proteus  bacilli  prepare  gelatin 
for  assimilation  through  the  action  of  a  proteolytic  ferment,  the 
ferment  is  not  elaborated  by  them  under  these  conditions.  A  pre- 
cisely similar  restriction  of  the  development  of  gelatin-liquefying 
ferments  by  utilizable  sugars  occurs  in  cultures  of  cholera  vibrios  and 
other  bacteria  which  habitually  liquefy  this  medium.  In  each  instance 
the  same  explanation  holds  true. 

4.  Diphtheria  bacilli  do  not  produce  their  characteristic  powerful 
extracellular  toxin  in  the  presence  of  utilizable  carbohydrate — dex- 
trose— as  Theobald  Smith1  showed  several  years  ago.     The  toxin  is 
only  formed  in  sugar-free  media.     In  this  case  again  the  dextrose 
shields  the  protein  of  culture  media  from  attack  by  the  diphtheria 
bacillus,  and  consequently  prevents  the   formation  of  toxin   which 
is  apparently  a  true  excretion  produced  incidental  to  the  utilization 
of  protein  for  energy  by  these  organisms.     Similarly,  tetanus  and 
Shiga  bacilli  fail  to  produce  toxin  in  the  presence  of  utilizable  carbo- 
hydrates. 

5.  Colon   and   proteus   bacilli   produce    considerable   amounts   of 
indol  in  sugar-free  media,  but  no  indol  in  the  same  media  to  which 
utilizable  sugar  has  been  added.     Here  again  the  carbohydrate  is 
attacked  by  these  organisms  in  preference  to  the  protein.    The  fol- 
lowing table  summarizes  briefly  the  salient  features  of  the  above 
discussion : 

Sugar-  free  media.2  Dextrose  media.2 

Nitrogen  substance,  Nitrogen  substance, 

Chemical  composition  of  bacteria.  per  cent.  per  cent. 

1.  Pfeiffer  bacillus 70.0  53.7 

Pneumo  bacillus 79 . 8  63 . 6 

Rhinoscleroma  bacillus    .  76 . 2  62 . 1 


2.  Diphtheria  bacillus 


3.  B.  tetani  .... 

B.  dysenterise  (Shiga) 

4.  B.  proteus 

Sp.  cholerse    . 

5.  B.  coli,  B.  proteus: 

Odor     .... 
Reaction    . 
Products    . 


Sugar-free  media. 
Powerful  extracellular  toxin 

of  which  on  the  average 

0.005  c.c.  kills  guinea-pigs. 
Powerful  extracellular  toxin 

produced. 
Toxin  present. 
Soluble,  extracellular  gela- 

tinase  formed. 
Soluble,  extracellular  gela- 

tinase  formed. 

Foul. 

Strongly  alkaline. 
H2S,    indol,    phenols,    am- 
monia, etc. 


Sugar  media. 

No  toxin  produced;  several 
cubic  centimeters  medium 
fails  to  kill  guinea-pigs. 

No  toxin  produced. 

No  toxin  present. 
No  gelatinase  formed. 

No  gelatinase  formed. 


None. 

Strongly  acid. 

H2,  CO2,  lactic  acid. 


1  Tr.  Assn.  Am.  Phys.,  1896. 

2  Nitrogenous  constituents  and  reaction  precisely  the  same  in  both  sugar-free  and 
sugar-containing  media.     The  only  difference  is  that  the  dextrose  medium  contains 
1  per  cent,  of  dextrose  in  addition.     The  organisms  studied  have,  therefore,  a  choice 
between  protein  and  sugar  for  catabolic  purposes. 


FERMENTATION  AND  PUTREFACTION  83 

DC.    FEEMENTATION  AND  PUTREFACTION. 

The  terms  "fermentation"  and  "putrefaction"  have  been  confused 
and  even  used  •  synonymously-  in  bacteriological,  chemical  and  even 
legal  nomenclature,  but  they  represent  essentially  distinct  and  generic 
types  of  bacterial  activity.  They  indicate,  or  should  indicate 
respectively,  microbic  decomposition  of  two  quite  distinct  types  of 
organic  compounds,  the  carbohydrates  and  closely  related  nitrogen- 
free  compounds,  on  the  one  hand  (fermentation),  and  nitrogenous 
organic  substances  on  the  other  hand,  putrefaction.  There  are 
substances  intermediate  in  character  between  carbohydrates  and 
proteins,  or  fats  and  nitrogen-containing  compounds  in  which  it 
would  be  difficult  to  predict  a  priori  which  term  would  be  correct 
— glucose  amine  is  such  a  substance.  Glucose  amine  is  an  amino- 
aldose,  containing  both  nitrogen  and  carbohydrate  groupings.  Such 
instances,  however,  are  uncommon  and  do  not  militate  against  the 
correctness  of  the  general  theory  that  fermentation  and  putrefaction 
are  distinct  processes.1 

Fischer2  has  defined  fermentation  in  the  broad  sense  it  should  be 
used  in  bacteriology,  essentially  in  the  following  terms:  "Fermenta- 
tion is  the  biochemical  decomposition  of  nitrogen-free  compounds, 
chiefly  carbohydrates,  by  the  action  of  microorganisms."  Similarly, 
putrefaction  is  defined  as  "The  biochemical  decomposition  of  nitro- 
genous organic  compounds  by  the  action  of  microorganisms." 

Fermentation  and  putrefaction  are  probably  enzyme  phenomena. 

Transposing  the  sparing  action  of  utilizable  carbohydrate  for 
protein,  which  has  been  repeatedly  emphasized  in  the  preceding 
pages,  it  may  be  stated  that  in  the  catabolic  phase  of  bacterial  metab- 
olism "fermentation  takes  precedence  over  putrefaction,"3  meaning 
by  that  that  bacteria  which  can  utilize  carbohydrate  derive  their 
energy  requirements  from  the  utilizable  carbohydrate  when  they  are 
growing  in  media  containing  both  carbohydrate  and  protein.  The 
results  of  this  sparing  action  of  utilizable  carbohydrate  for  protein 
have  been  indicated  in  the  preceding  pages,  sections  V-VIII,  inclusive. 

1  Kendall,  Jour.  Med.  Research,  1911,  N.  S.,  xx,  140-144. 

2  Vorlesungen  iiber  Bakterien,  1903,  II  Aufl.,  206. 

3  Kendall,  Jour.  Med.  Research,  1911,  N.  S.,  xx,  140-144. 


CHAPTER  V. 


SAPROPHYTISM,  PARASITISM,  AND  PATHOGENISM. 


I.  DEFINITIONS  AND  LIMITS. 
II.  THE  CYCLE  OF  PARASITISM. 

III.  THE  CYCLE  OF  PATHOGENISM. 

IV.  DISTRIBUTION   OF   PARASITIC   AND 

PATHOGENIC   BACTERIA   IN    NA- 
TURE. 

V.  How  PARASITIC  AND  PATHOGENIC 
BACTERIA  REACH  MAN. 

A.  The  Occurrence  of  Parasitic 

Bacteria  upon  the  Bodies  of 
Healthy  Men  and  Animals. 

B.  How      Pathogenic      Bacteria 

Reach  the  Body. 

1.  Air-borne  Infection. 

(a)  Dust. 
(6)  Droplet. 

2.  Soil-borne  Infection. 

3.  Water-borne  Infection. 

4.  Food-borne  Infection. 

5.  Animal  Carriers. 

(a)  Direct  Contact. 
(6)  Indirect  Transfer. 

(c)  Mechanical 

Transfer. 

(d)  Intermediary  Host. 


6.  Human  Carriers. 

7.  Contact  Infection. 

8.  Germinal    and    Prenatal 

Infection. 

C.  Portal  of  Entry:  Atria  of  In- 

vasion. 

1.  Skin  and  Adnexa:  Ear, 

Eye.  SubcutaneousTis- 
sue,  Tonsils,  Salivary 
Glands,  Nasal  Cavity, 
Lungs. 

2.  Mucous     Membranes: 

Mouth,  Stomach,  In- 
testines. 

3.  Geni to-urinary    System: 

Vagina,  Uterus,  Ure- 
thra, Urinary  Bladder 
and  Ureter,  Kidneys. 

D.  Where  Bacteria  Multiply  in 

the  Body. 

E.  Where    and     How     Bacteria 

Escape  from  the  Body. 
VI.  BALANCED  PATHOGENISM;  EPIDEMI- 
OLOGY. 


I.     DEFINITIONS   AND   LIMITS. 

THE  most  conspicuous  and  important  function  of  bacteria  in  the 
economy  of  Nature  is  to  maintain  a  continuity  between  the  Animal 
and  Vegetable  Kingdoms  by  restoring  in  utilizable  form  to  the  Plant 
World  the  elements  contained  in  the  complex  organic  compounds 
which  comprise  the  dead  bodies  of  plants,  animals  and  their  products. 
Bacteria  dissipate  much  of  the  energy  accumulated  in  these  dead 
bodies  and  oxidize  the  elements  contained  in  them  to  inorganic,  fully 
mineralized  salts.  These  salts  are  resynthesized  by  the  chlorophyll- 
bearing  plants  through  the  energy  of  sunlight  to  carbohydrates, 
proteins  and  fats,  and  in  these  complex  combinations  the  elements 
are  again  available  for  animal  food. 

The  bacteria  which  live  upon  this  dead  organic  matter,  and  whose 
function  it  is  to  effect  its  degradation  and  ultimate  mineralization, 
are  called  saprophytic  bacteria.  They  are  specifically  the  most 


DEFINITIONS  AND  LIMITS  85 

numerous,  chemically  the  most  active,  and  economically  the  most 
important  members  of  the  phylum  Bacteriacese.  They  are  rarely 
pathogenic,  that  is,  they  rarely  initiate  disease  in  man  or  the  lower 
animals.  Whenever  they  are  found  associated  with  morbid  processes 
their  presence  is  usually  to  be  explained  on  the  ground  that  they  are 
secondary  invaders. 

A  smaller  group  of  bacteria  are  parasitic,  that  is,  they  exist  upon 
the  bodies  of  living  plants,  animals  or  men.  Many  of  them  are  rarely 
met  with  in  Nature  far  removed  from  their  respective  hosts.  Their 
activities  are  not  usually  in  opposition  to  those  of  their  host  and  their 
presence  is  therefore  unnoticed.  They  may  become  invasive,  how- 
ever, whenever  the  natural  barriers,  which  ordinarily  suffice  to  keep 
them  out,  are  impaired. 

From  the  parasitic  bacteria  there  has  been  gradually  evolved  a 
small  but  formidable  group  of  organisms,  the  pathogenic  bacteria, 
whose  activities  are  in  partial  opposition  to  those  of  their  host.  The 
pathogenic  bacteria,  like  the  parasitic  bacteria,  require  a  living  host, 
but  they  differ  from  the  parasitic  forms  in  that  they  actually  invade 
their  hosts  and  induce  progressive  disease  from  host  to  host. 

There  are  no  sharply  definable  limits  between  these  three  groups 
of  bacteria,  the  saprophytic,  parasitic,  and  pathogenic;  the  latter 
appear  to  have  arisen  from  the  former  by  a  process  of  evolution. 
Certain  general  modifications  in  the  general  types  of  chemical  activity 
manifested  by  these  groups  are  discernible,  however,  which  are  partly 
the  result  and  partly  the  cause  of  their  change  in  environment  as 
they  have  passed  from  a  saprophytic  to  a  parasitic  existence.  Promi- 
nent among  these  modifications  and  activities  is  a  gradual  decrease 
in  the  intensity  with  which  the  parasitic  and  pathogenic  bacteria  act 
upon  their  environment. 

The  essential  function  of  the  saprophytic  bacteria  in  Nature  is  to 
effect  a  rapid,  deep-seated  degradation  of  organic  matter  to  simple 
compounds;  these  organisms  decompose  a  relatively  large  amount 
of  substance  in  a  relatively  short  time.  They  are  chemically  active 
and  many  of  them  form  highly  resistant  spores  which  enable  them 
to  survive  prolonged  periods  of  environmental  vicissitude.  The 
habitually  parasitic  bacteria,  on  the  other  hand,  which  exist  upon 
the  bodies  of  living  animals,  and  the  progressively  pathogenic  bacteria 
which  develop  within  the  tissues  of  animals  are  not  subjected  to 
extremes  of  temperature  and  food  supply;  they  rarely  or  never  form 
spores.  The  chemical  activity  of  these  organisms  is  usually  much 


86         SAPROPHYTISM,   PARASITISM,  AND  PATHOGENISM 

less  pronounced  than  that  of  the  saprophytic  bacteria.1  Indeed, 
intense  chemical  activity  would  be  incompatible  with  their  continued 
parasitic  existence,  for  the  damage  to  their  host  would  be  insupport- 
able. The  parasitic  and  pathogenic  bacteria  do  not,  for  example, 
produce  widespread  liquefaction  of  the  tissues,  even  when  large 
numbers  of  them  are  actually  growing  in  the  body  of  the  host.  The 
growth  of  invasive  organisms  in  the  animal  body  is  characterized  by 
subtle  changes  in  the  composition  of  the  tissues  of  the  host  and  the 
development  of  these  reciprocal  reactions  between  host  and  parasite, 
which  collectively  are  included  in  the  newly  developed  science  of 
Immunology. 

It  would  appear,  therefore,  that  in  their  evolution  toward  parasit- 
ism, those  bacteria  which  could  thrive  without  producing  deep-seated 
and  rapid  degradation  of  proteins,  that  is  to  say,  whose  metabolism 
approached  more  closely  the  intracellular  metabolism  of  their  host, 
would  be  the  more  adaptable  to  a  parasitic  existence,  and  this  is  in 
accord  with  what  is  known  of  the  chemistry  of  these  organisms. 
Their  metabolism  approaches  rather  closely  that  of  their  host. 

H.     THE   CYCLE   OF   PARASITISM. 

The  cycle  of  parasitism  for  bacteria  whose  life  cycle  is  such  that 
but  a  limited  excursion  outside  their  host  is  possible  for  them — and 
this  appears  to  be  the  case  for  the  majority  of  organisms  parasitic  on 
man — consists  of  three  separate  and  well-defined  stages,  as  Theobald 
Smith2  has  so  clearly  pointed  out.  They  must — first — reach  an  appro- 
priate host;  secondly — multiply  at  least  temporarily  thereon,  and 
thirdly — escape  to  other  suitable  hosts.  Each  phase  of  this  parasitic 
existence  must  be  exactly  fulfilled,  otherwise  the  cycle  is  broken  and 
that  particular  strain  dies  out.  It  is  not  surprising,  therefore,  that 
the  bacteria  habitually  parasitic  for  man  are  found  variously  upon 
the  surface  of  the  body — in  the  upper  respiratory  tract,  the  gastro- 
intestinal tract,  or  upon  the  mucous  surfaces  which  are  in  direct 
communication  with  the  exterior.  Escape  from  the  body  of  the  host 
to  other  hosts  is  readily  accomplished  from  these  positions.3 

Under  special  conditions,  parasitic  bacteria  may  actually  invade 
the  body  of  the  host  and  become,  therefore,  temporarily  pathogenic. 

1  Theobald  Smith,  Am.  Med.,  October  22,  1904,  viii;    Kendall,  Boston  Med.  and 
Surg.  Jour.,  1913,  clxix,  749. 

2  Theobald  Smith,  loc.  cit. 

3  Theobald  Smith,  loc.  cit. 


THE  CYCLE  OF  PATHOGEN  ISM  87 

Such  an  invasion  is  usually  subsequent  to  a  preexisting  disease  or  to 
local  weakening  of  the  tissues  which  under  normal  conditions  suffice 
to  exclude  these  organisms.  The  disease  produced  by  parasitic  organ- 
isms is  usually  non-specific  in  character  and  sporadic  in  distribution, 
and  ordinarily  it  does  not  attain  epidemic  proportions.  The  bacteria 
which  have  penetrated  into  the  tissues  of  the  host  are  locked  up  there, 
as  it  were,  and  their  descendants  cannot  escape  to  other  hosts,  at  least, 
in  numbers  sufficient  to  perpetuate  the  invasive  strain,  for  these 
organisms  have  not  perfected  their  pathogenic  cycle.  Parasitic 
organisms,  in  other  words,  are  "  opportunists,"  as  Theobald  Smith 
has  admirably  called  them,  rarely  initiating  disease,  but  usually  able 
to  penetrate  the  body  as  secondary  or  terminal  invaders.  The  colon 
bacillus,  for  example,  is  an  habitual  parasite  in  the  gastro-intestinal 
tract  of  man  and  many  animals.  Under  certain  conditions  it  may 
become  invasive,  causing  cystitis,  appendicitis,  peritonitis,  or  other 
inflammatory  lesions,  but  it  does  not  ordinarily  become  progressively 
pathogenic  for  successive  hosts,  producing  epidemics  of  cystitis,  ap- 
pendicitis or  peritonitis.  The  staphylococcus  is  a  common  inhabitant 
of  the  skin  of  healthy  man.  When  the  continuity  of  the  epidermis  is 
destroyed,  the  organism  may  become  invasive,  causing  furuncles, 
osteomyelitis,  or  endocarditis.  The  pneumococcus  is  found  in  the 
respiratory  tract  of  many  normal  men,  particularly  in  large  cities, 
where  it  exists  as  an  "opportunist,"  ordinarily  producing  no  harmful 
effects,  but  frequently  becoming  invasive  and  producing  a  variety  of 
lesions  when  the  general  resistance  of  the  host  is  lowered.1  These 
parasitic  bacteria  have  not  perfected  their  mechanism  of  entry  into 
the  tissues  of  the  host,  and  of  escape  from  the  tissues  to  the  exterior, 
consequently  those  strains  which  accidentally  become  invasive  are 
locked  up  in  the  body  and,  as  a  rule,  either  are  overwhelmed  by 
thjeir  host  or  perish  with  it.  They  are  imperfectly  pathogenic,  in  other 
words. 

m.     THE  CYCLE  OF  PATHOGENISM. 

Habitually  pathogenic  bacteria — those  organisms  which  produce 
progressive,  specific  disease  from  host  to  host — actually  invade  the 
living  bodies  of  animals  or  man.  This  invasion  may  be  direct*  in  which 
event  the  microorganisms  actually  enter  the  tissues  or  body  fluids 

1  Recent  studies  by  Cole  and  his  associates  indicate  that  the  ordinary  "mou^" 
pneumococcus  differs  serologically  from  the  strains  found  in  the  saliva  of  pneumonl^ 
cases.  It  is  not  improbable  that  similar  serological  differences  may  be  demonstrated 
in  the  group  of  the  streptococci. 


88         SAPROPHYTISM,  PARASITISM,  AND  PATHOGENISM 

and  multiply  there,  or  it  may  be  indirect,  in  which  instance  their  soluble 
toxins  alone  are  absorbed  by  the  host.  The  cycle  of  pathogenism, 
therefore,  is  more  complex  than  the  cycle  of  parasitism ;  it  necessitates 
lodgement  of  the  invading  microbe  on  the  body  of  the  host,  the  location 
and  penetration  of  the  necessary  portal  of  entry  (which  involves  an 
initial  skirmish  between  the  organism  and  the  non-specific  natural 
defences  of  the  host),  growth  within  the  tissues  of  the  host  in  the 
presence  of  opposition  there,  escape  from  the  tissues  to  the  surfaces 
of  the  host  or  to  some  channel  in  communication  with  the  exterior 
and,  finally,  the  transmission  of  the  organism,  directly  or  indirectly, 
to  other  suitable  hosts.  If  the  organism  cannot  force  an  entrance  to 
the  tissues  of  the  host,  that  is,  if  the  natural  defences  of  the  host 
suffice  to  keep  out  the  prospective  invader,  the  latter  usually  perishes 
and  no  infection  takes  place;  if  the  organism  does  penetrate  the  tissues 
of  the  body,  the  invasion  and  growth  of  the  microorganism  leads  to 
disturbances  of  structure,  function  or  composition  of  the  host,  which 
are  abnormal  and  inimical  to  his  well-being.  The  production  of  disease, 
therefore,  depends  ordinarily  upon  the  ability  of  the  microorganism 
to  multiply  in  the  tissues  or  the  body  fluids  of  the  host;  bacteria  which 
cannot  force  an  entrance  into  the  tissues  of  the  host,  multiply  there 
and  escape  to  the  exterior  and  eventually  to  other  susceptible  hosts 
do  not  produce  progressive  disease. 

The  nature  and  extent  of  the  disease  produced  depends  upon  several 
factors:  (1)  the  kind  of  microorganism;  (2)  the  number  of  micro- 
organisms; (3)  their  ability  to  locate  and  force  an  entrance  to  the 
tissues  of  the  body  (their  virulence,  in  other  words) ;  (4)  the  location 
and  extent  of  their  multiplication  in  the  tissues  of  the  host;  (5)  the 
response  of  the  tissues  of  the  host  to  this  invasion,  and  (6)  the  nature 
and  extent  of  the  secondary,  specific  defense  of  the  host  in  response  to 
the  invasion. 

The  contagiousness  of  a  disease  depends  upon  the  ability  of  the 
invading  organisms  to  escape  from  their  host  in  sufficient  numbers  to 
infect  new  hosts  and  to  survive  environmental  vicissitudes  until  new 
hosts  are  reached.  A  few  examples  will  indicate  the  principal  vari- 
ants of  the  pathogenic  cycle  commonly  met  with  among  progressively 
pathogenic  bacteria. 

The  tubercle  bacillus  ordinarily  gains  entrance  to  the  host  through 
the  air  passages.  The  organisms  pass  through  the  alveoli  of  the  lungs, 
set  up  infection  there,  and  gradually  are  shut  off  from  communication 
with  the  exterior  through  the  formation  of  the  tubercle.  After  a 


DISTRIBUTION  OF  PARASITIC  AND  PATHOGENIC  BACTERIA     89 

longer  or  shorter  time,  these  tubercles  eventually  break  down,  typically 
into  the  air  passages — and  discharge  there  large  numbers  of  tubercle 
bacilli.  These  are  coughed  up  by  the  patient  and  are  eliminated  from 
the  body,  usually  in  enormous  numbers,  by  droplets  and  in  the  sputum. 
Pulmonary  tuberculosis  is  typically  a  chronic,  focal  disease.  The 
perpetuation  of  the  tubercle  bacillus  is  assured  through  their  elimina- 
tion from  the  diseased  body  in  enormous  numbers  through  long  periods 
of  time,  their  ability  to  resist  desiccation,  and  the  relative  directness 
with  which  they  reach  other  hosts. 

The  typhoid  bacillus  gains  entrance  to  the  body  through  the  mouth 
and  the  intestinal  tract.  The  organisms  penetrate  the  intestinal 
mucosa,  develop  in  the  internal  organs,  particularly  the  spleen,  and 
after  a  rather  definite  excursion  in  the  tissues  of  the*  body,  enter  into 
the  intestinal  tract  again,  either  through  ulcers  or  the  gall-bladder  or, 
occasionally,  they  appear  in  the  urine.  They  are  eliminated  from  the 
body  in  great  numbers,  either  with  the  feces,  or  less  commonly,  the 
urine,  and  they  gain  access  immediately  to  other  subjects  through 
direct  contact  or  more  or  less  indirectly  through  water  or  food,  in 
sufficient  numbers  to  set  up  infection  in  at  least  some  of  them. 

The  gonococcus  is  transmitted  directly  by  contact.  Occasionally 
the  infection  may  be  somewhat  less  direct,  involving  the  conjunctiva. 

The  plague  bacillus  may  be  transmitted  from  host  to  host,. either 
directly  in  the  case  of  pneumonic  plague,  where  great  numbers  of 
plague  bacilli  are  coughed  up  from  the  lungs  of  one  patient  and  trans- 
mitted through  inhalation  to  other  patients,  or  somewhat  more 
indirectly,  as  is  the  case  in  bubonic  plague.  Bubonic  plague  appears  to 
be  a  true  septicemia;  the  plague  bacilli  circulate,  at  least  temporarily, 
in  the  blood,  and  they  are  removed  from  the  blood  of  one  patient  and 
transmitted  to  another  patient  (either  man  or  rat)  through  the  agency 
of  the  flea,  which  acts  potentially  as  an  hypodermic  syringe,  as  it  were, 
in  this  instance.  Plague  bacilli  are  locked  up  in  the  tissues  of  the  host 
and  were  it  not  for  the  agency  of  a  suctorial  insect,  as  the  flea,  bubonic 
plague  would  almost  certainly  disappear,  because  the  organisms  have 
not  perfected  for  themselves  any  mechanism  of  escape  from  one  V)st 
to  the  other. 

IV.    DISTRIBUTION  OF  PARASITIC  AND  PATHOGENIC  BACTERIA 

IN  NATURE. 

It  has  been  shown  in  previous  sections  that  comparatively  few,  if 
indeed  any,  of  those  bacteria  habitually  parasitic  or  pathogenic  for 


90         SAPROPHYTISM,  PARASITISM,  AND  PATHOGENISM 

man  are  found  in  Nature  far  removed  from  rather  intimate  association 
with  their  hosts.  This  is  in  accordance  with  the  fact  that  few,  if  any, 
of  these  organisms  are  provided  with  spores  which  would  enable  them 
to  survive  exposure  to  long  periods  of  conditions  unfavorable  to  their 
growth.  It  is  true,  however,  that  some,  at  least,  of  these  organisms, 
as  for  example,  the  typhoid  bacillus,  can  survive  for  longer  or  shorter 
periods  of  time  in  the  soil,  particularly  if  it  be  frozen,  or  in  water,  for 
days  or  even  weeks.  There  is  little  evidence  that  these  bacteria  multiply 
extensively  outside  the  body;  on  the  contrary,  they  tend  to  die  off 
rather  rapidly.  In  any  event,  their  existence  depends  upon  their 
reaching  a  suitable  host  again  within  a  comparatively  brief  period. 

There-  are  a  few  spore-forming  bacteria  which  occasionally  infect 
man  when  associated  conditions  are  favorable  for  them.  Of  these 
the  bacillus  of  lockjaw,  B.  tetani;  of  botulism,  B.  botulinus;  the 
gas  bacillus,  B.  aerogenes  capsulatus;  and  the  anthrax  bacillus  are 
well-known.  These  organisms  are  not  habitual  parasites,  however; 
they  are  "saprophytic  opportunists."  That  is,  they  could  in  all 
probability  exist  if  man  were  eliminated  from  their  environment. 

V.    HOW  PARASITIC  AND  PATHOGENIC  BACTERIA  REACH  MAN. 

A.  The  Occurrence  of  Parasitic  Bacteria  upon  the  Bodies  of  Healthy 
Men  and  Animals. — The  continual  exposure  of  the  skin  of  man  to  his 
environment  makes  it  almost  inevitable  that  microbes  shall  collect 
there.  It  is  quite  probable,  however,  that  the  large  number  of  micro- 
organisms which  reach  the  skin  are  not  only  non-pathogenic,  they  are  not 
even  habitually  parasitic.  Most  of  them  are  found  there  only  trans- 
iently. Certain  organisms,  however,  occur  among  these  adventitious 
microbes,  which  appear  to  be  habitual  parasites,  and  many  of  these 
bacteria,  under  certain  conditions,  produce  disease.  Of  these,  Staphy- 
lococcus  aureus  and  albus  and  Streptococcus  pyogenes  are  almost 
invariably  present  not  only  on  the  skin,  but  on  the  exposed  mucous 
membranes,  particularly  those  of  the  nose  and  throat.  The  influenza 
bacillus,  diphtheria  bacillus,  the  pneumococcus,  and  even  the  tubercle 
bacillus,  meningococcus  and  other  organisms  may  also  be  occasionally 
found,  particularly  in  the  nose  and  throat  of  healthy  men.  The 
occurrence  of  these  organisms  is  readily  explained;  the  secretions  of 
the  nose  and  throat,  as  well  as  that  of  the  skin  are  excellent  culture 
media  for  these  organisms,  which  collect  at  these  sites  and  grow  upon 
the  various  secretions  and  desquamated  cells. 


PARASITIC  AND  PATHOGENIC  BACTERIA  91 

The  majority  of  these  organisms,  however,  particularly  the  coccal 
forms,  as  the  staphylococcus,  streptococcus  and  pneumococcus  are  to 
be  regarded  as  "opportunists";  they  do  not  of  themselves  initiate 
disease,  as  a  rule.  They  are  to  be  regarded  rather,  as  Theobald  Smith 
has  called  them,  "organisms  of  the  diseased  state/'  because  of  their 
invasion  of  the  bojdy  secondary  to  other,  intercurrent  diseases.  Even 
the  tubercle  bacillus  and  the  diphtheria  bacillus,  particularly  the 
latter,  have  been  found  in  the  mouths  of  men  who  apparently  have 
had  neither  tuberculosis  nor  diphtheria,  yet  these  organisms  appear 
to  be  virulent  when  tested  in  the  usual  manner  and  presumably  might 
be  able  to  incite  disease  whenever  conditions  favor  their  entrance  to 
the  tissues  of  the  body.  Theoretically  at  least,  people  who  harbor 
these  organisms  are  potential  sources  of  danger  to  others.  Even  the. 
internal  organs  of  healthy  individuals  may  contain  parasitic  bacteria 
without  harm,  although  these  organisms  naturally  are  not  present  in 
large  numbers.  Tubercle  bacilli  have  been  found  occasionally  in 
lymph  glands  in  normal  man  and  in  cattle.  Intestinal  bacteria  also 
occur  not  infrequently  in  the  apparently  healthy  tissues  of  the  body. 
In  rare  instances,  B.  coli  may  be  present  in  the  urinary  bladder  without 
causing  noteworthy  symptoms. 

B.  How  Pathogenic  Bacteria  Reach  the  Body. — The  manner  in 
which  bacteria  of  the  "opportunist"  type  reach  the  body  has  been 
considered  above.  It  is  now  necessary  to  consider  the  manner  in  which 
bacteria  which  cause  progressive  disease  from  man  to  man  reach 
the  body. 

1.  Air-borne  Infection. — Bacteria  which  cause  progressive  disease, 
particularly  of  the  respiratory  tract,  are  discharged  from  the  diseased 
body  principally  through  the  mouth  and  nose  and  find  lodgment  in  the 
environment  of  the  patient  through  the  medium  of  the  air,  from  whence 
they  settle  upon  various  substances,  as  food,  clothing,  and  walls  and 
floors  of  rooms.  These  bacteria  probably  do  not  proliferate  to  any 
extent  outside  of  the  body,  but  they  resist  drying  and  may  remain 
fully  virulent  for  considerable  periods  of  time  and  potentially  able  to 
infect  a  certain  proportion  of  those  individuals  who  may  be  exposed 
to  them. 

These  air-borne  infections  are  transmitted  in  at  least  two  rather 
distinct  ways:  (a)  by  dust,  and  (6)  by  droplet  infection. 

(a)  Organisms  which  are  transmissible  through  dust  must  first 
of  all  be  able  to  survive  considerable  periods  of  drying.  The  larger 
particles  of  dust  to  which  bacteria  may  become  attached  soon  settle 


92         SAPROPHYTISM,   PARASITISM,  AND  PATHOGENISM 

from  the  air,  but  smaller  particles  may  remain  suspended  for  some 
time,  depending  on  the  velocity  of  air  currents  and  the  nature,  size 
and  shape  of  the  particles.  Dusting  and  sweeping  in  rooms  naturally 
stir  up  particles  which  have  settled  from  the  air,  and  even  larger  par- 
ticles may  be  resuspended  in  this  way.  Tuberculosis  has  frequently 
been  suspected  to  have  been  transmitted  through  the  inhalation  of 
infected  dust  particles,  that  is,  particles  of  dust  which  have  dried 
tubercle  bacilli  adhering  to  them.  Careful  investigation  has  shown  that 
houses  in  which  careless  consumptive  patients  have  lived  have  been 
responsible  for  the  transmission  of  tuberculosis.  The  ward-room  of 
a  battle  ship  is  known  to  have  become  infected  with  tubercle  bacilli 
early  in  its  career  and  at  least  two  successive  details  of  officers  con- 
tracted tuberculosis  in  this  place.  Guinea-pigs  exposed  on  the  floor 
of  these  so-called  tuberculous  rooms  are  quite  frequently  successfully 
infected  with  the  tubercle  bacillus. 

The  extent  to  which  dust  dissemination  is  a  factor  in  transmitting 
disease,  however,  is  not  at  all  definitely  known.  It  must  be  emphasized 
that  the  transmission  of  disease  through  dust  is  not  necessarily  a 
very  direct  one,  because  the  inciting  organisms  may  pass  a  very  con- 
siderable period  of  time  in  dust  before  they  reach  a  favorable  host. 
In  this  sense,  transmission  of  disease  by  dust  is  a  relatively  latent  one. 

(6)  Droplet  Infection. — Fliigge1  and  his  pupils  were  the  first  to 
demonstrate  that  minute  droplets  of  spray  may  be  eliminated  from  the 
mouth  during  talking,  sneezing  and  coughing.  These  droplets  are 
frequently  carried  through  the  air  for  some  distance,  even  as  much  as 
ten  meters  in  a  quiet  room.  Usually  the  more  minute  particles  remain 
suspended  in  the  air  for  some  time.  The  possibility  of  droplet  infection 
has  been  definitely  proven  in  the  following  manner:  Agar  plates 
containing  sodium  carbonate  are  placed  at  various  heights  and  dis- 
tances from  the  experimenter,  who  places  in  his  mouth  a  solution  of 
phenolphthalein  and  then  talks  in  a  natural  manner,  expelling  droplets 
containing  phenolphthalein  during  his  speech.  This  dye  is  transmitted 
with  the  droplets  until  they  reach  the  agar  plates,  where  bright  red 
spots  are  produced  which  are  very  readily  observed.  In  like  manner, 
cultures  of  B.  prodigiosus  placed  in  the  mouth  will  infect  agar  plates 
at  similar  distances. 

The  transmission  of  disease  by  droplet  infection  may  be,  and  fre- 
quently is,  a  very  direct  one.  Bacteria  which  are  air-borne  or  borne 
by  droplets  may  remain  alive  for  several  weeks  in  indirect  sunlight, 

1  Ztschr.  f.  Hyg.,  1897,  xxv,  179. 


PARASITIC  AND  PATHOGENIC  BACTERIA  .       93 

but  all  of  them  are  readily  killed  if  they  are  exposed  to  direct  sunlight. 
The  virus  of  whooping-cough,  mumps,  measles,  influenza,  cerebro- 
spinal  meningitis,  pneumonic  plague,  tuberculosis,  the  exanthemata, 
the  diphtheria  bacillus,  and  possibly  the  pneumococcus  may  be  spread 
in  this  manner.  Air-borne  infections  probably  rarely  take  place 
in  the  open  air  where  the  sunlight  is  strong.  This  does  not  apply 
to  droplet  infections  where  one  individual  coughs,  talks  or  sneezes 
directly  into  the  face  of  another.  Air-borne  infections,  particularly 
droplet  infections,  are  potentially  common  where  overcrowding  occurs, 
as  in  tenements,  public  gatherings,  railway  trains,  schools,  and  factories. 

2.  Soil-borne   Infections. — Those    bacteria    which    are    occasionally 
pathogenic  for  man  and  produce  sporadic  disease  in  man,  and  whose 
habitat  is  the  soil,  are  for  the  most  part  spore-forming  organisms.    They 
commonly  enter  the  body  through  wounds.    Of  these  the  bacillus  of 
tetanus,  malignant  edema,  symptomatic  anthrax,  of  anthrax,  and  the 
gas  bacillus  are  the  best  known  but,  with  the  exception  of  the  latter, 
they  are  not  habitually  human  parasites.     Of  those  bacteria  which 
are  habitually   pathogenic   for  man,  typhoid,  cholera,  paratyphoid 
and  probably  dysentery  may  be  soil-borne,  but  ordinarily  infection 
with  these  organisms  does  not  take  place  through  the  soil. 

3.  Water-borne  Infection. — The  viruses  of  excrementitious  diseases — 
typhoid,  paratyphoid,  dysentery,  and  cholera — are  not  infrequently 
transmitted  from  man  to  man  through  contaminated  water.    Feces 
containing  these  organisms  get  into  water  supplies,  reach  man  again, 
incite  disease  in  man,  again  escape  in  the  feces  and  reenter  water 
courses,  thus  being  recirculated.    The  cycle  may  be  somewhat  more 
complex,  as  for  example,  when  typhoid  dejecta  are  thrown  upon  the 
ground  and  are  eventually  washed  directly  into  water  supplies  and 
thus  reach  man  again. 

4.  Food-borne    Infection. — A    considerable    number    of    pathogenic 
bacteria  may  reach  man  through  food,  although  food  which  is  infected 
is  usually  rendered  so  through  the  handling  of  it  by  man.     Milk  is 
probably  the  most  common  food  thus  to  be  infected  and  it  is  par- 
ticularly dangerous  for  two  reasons.     In  the  first  place,  its  opacity 
makes  it  difficult  to  distinguish  foreign  substances  which  may  be  in  it; 
and  again,  it  contains  all  the  elements  which  are  necessary  for  the 
food  of  man  and  incidentally  for  the  majority  of  bacteria.     Scarlet 
fever,  diphtheria,  tuberculosis  both  human  and  bovine,  Malta  fever, 
epidemic  sore  throat  or  tonsillitis,  typhoid,  dysentery,  foot-and-mouth 
disease,  many  diarrheas  of  children,  milk  sickness,  and  the  organisms 


94         SAPROPHYTISM,  PARASITISM,  AND  PATHOGENISM 

of  cholera  infantum,  and,  rarely,  Asiatic  cholera  as  well,  all  may  be 
transmitted  from  milk. 

Shell-fish,  particularly  oysters,  have  been  known  to  transmit  enteric 
diseases.  This  has  been  due,  in  the  past,  largely  to  their  exposure 
in  the  estuaries  of  rivers  where  sewage  flowed  freely  over  them. 
Typhoid  bacilli  enter  the  mantle  cavity  of  the  shell-fish,  remain  alive 
there  and  enter  the  digestive  tract  in  a  viable  state  when  the  shell- 
fish are  consumed  in  an  uncooked  condition. 

Meats,  particularly  from  beef  and  swine,  have  been  known  to  transmit 
paratyphoid  fever,  botulismus  (sausage  poisoning)  and  meat-poisoning 
as  well.  There  is,  in  addition,  a  group  of  cases  with  somewhat  insidious 
symptoms,  which  are  probably  due  to  the  consumption  of  food,  par- 
ticularly meat,  which  has  been  decomposed  by  saprophytic  bacteria. 

5.  Animal  Carriers. — The  microbic  diseases  which  are  transmissible 
to  man  from  animals  and  from  man  to  man  by  animals  are  varied 
in  character.  They  comprise  protozoan  and  bacterial  infections  and 
the  so-called  "  filterable  viruses."  Of  these  diseases,  comparatively 
few  are  common  to  man  and  animals.  Microorganisms  may  be  trans- 
mitted to  man  by  animal  carriers  in  at  least  four  distinct  ways: 

(a)  By  direct  contact. 

(b)  By  indirect  transfer. 

(c)  By  mechanical  transfer. 

(d)  By  intermediary  hosts. 

(a)  Direct  Contact. — The  transfer  of  glanders  from  the  horse  to 
man,  of  anthrax  from  cattle  and  sheep  to  man  and  of  hydrophobia 
from  dogs  to  man  represents  direct  transfer  of  the  virus  from  the  sick 
animal  to  the  well  man.    Other  diseases  are  thus  transmitted,  but  the 
examples  given  suffice  for  illustration. 

(b)  Indirect  Transfer. — Insects  are  common  carriers  in  the  indirect 
transmission  of  the  virus  of  disease  from  man  to  man.    Flies  are  known 
to  have  carried  typhoid  bacilli  from  typhoid  dejecta  to  milk  or  other 
food,  which  in  turn  has  been  consumed  by  man,  resulting  in  infection. 
The  same  insect,  doubtless,  when  conditions  are  favorable,  can  and 
does  carry  other  enteric  bacteria — paratyphoid,  dysentery  and  even 
cholera  organisms.     It  is  very  probable  that  other  insects  also  par- 
ticipate in  the  indirect  transmission  of  bacteria.    Acute  conjunctivitis, 
particularly  that  form  which  is  prevalent  in  Egypt,  is  supposed  to 
be  spread  in  this  manner. 

(c)  Mechanical  Transfer. — Suctorial  insects  are  known  to  transmit 
the  viruses  of  certain  diseases  which  circulate  in  the  blood  stream  of 


PARASITIC  AND  PATHOGENIC  BACTERIA  95 

animals  to  man,  incidental  to  feeding.  Thus,  the  flea  transmits  the 
plague  bacillus  from  rat  to  man,  from  man  to  man,  and  possibly  from 
man  to  the  rat.  The  louse  similarly  spreads  the  virus  of  typhus  from 
man  to  man.  In  the  instances  cited  the  insect  is  probably  not  a  true 
intermediary  host,  for  the  virus  does  not  necessarily  multiply  in  the 
insect,  nor  does  the  virus  undergo  any  essential  transformation,  so 
far  as  is  known,  in  the  insect.  Nevertheless,  the  transmission  of  the 
viruses  of  these  diseases — bubonic  plague  and  septicemia,  for  example, 
depends  upon  the  agency  of  suctorial  insects  for  their  passage  from 
host  to  host.  Other  insects  also  transmit  disease,  but  the  evidence 
in  a  majority  of  instances  is  somewhat  less  definite  than  the  cases 
cited. 

(d)  Intermediary  Hosts. — Certain  insects,  notably  mosquitoes, 
transmit  disease  from  man  to  man  only  after  the  virus  has  passed 
an  extracorporeal  cycle  in  the  extrinsic  host — the  mosquito  in  this 
instance.  Thus,  Anopheles  transmits  malaria  from  man  to  man  and 
Stegomyia  fasciata,  or  as  it  is  now  called,  Aedes  calopus,  transmits  in 
similar  manner,  the  virus  of  yellow  fever.  Transmission  in  these  cases 
is  through  the  female  insect  and  a  definite  interval  (latent  period) 
must  elapse  between  the  time  of  biting  the  patient  and  the  time  when 
the  mosquito  becomes  infective  to  the  non-immune  host. 

6.  Human  Carrriers. — Individuals  who  are  apparently  healthy 
occasionally  harbor  within  their  bodies  (in  free  communication  with 
the  exterior,  however,  either  through  the  respiratory  tract,  the  gastro- 
intestinal tract,  the  urinary  tract  or  the  skin)  bacteria  which  are 
capable  of  inciting  disease  in  others.  Such  individuals  are  known  as 
bacillus  carriers;  frequently  they  eliminate  these  pathogenic  bacteria 
in  large  numbers. 

The  bacillus  carrier  may  or  may  not  give  a  history  indicating 
recovery  from  an  infection  of  the  specific  organism  which  he  "carries." 
Bacillus  carriers  may  be  temporary  carriers,  in  which  event  they  harbor 
the  pathogenic  bacteria  for  but  a  few  weeks;  or  they  may  become 
habitual  carriers,  in  which  case  the  organism  may  be  excreted  for 
considerable  periods  of  time,  even  years.  The  excretion  may  be 
constant  or  intermittent. 

The  typhoid  bacillus  is  a  common  organism  to  be  thus  carried.  It 
appears  to  localize  eventually  in  the  gall-bladder  or  the  bile  ducts,  less 
commonly  in  the  urinary  bladder,  and  it  may  appear  occasionally 
in  large  numbers  in  the  feces  or  urine  of  the  carrier.  Women  are  more 
commonly  found  to  be  typhoid  carriers  than  men.  Similarly,  para- 


96         SAPROPHYTISM,  PARASITISM,  AND  PATHOGENISM 

typhoid,  dysentery  and  cholera  organisms  may  be  excreted  in  the  feces 
through  long  periods  of  time,  rarely  or  never,  however,  in  the  urine. 
Slowly  progressing  focal  diseases,  as  pulmonary  tuberculosis  are, 
in  a  sense,  spread  by  carriers,  for  the  patient  may  survive  for  years, 
excreting  daily  large  numbers  of  tubercle  bacilli.  The  line  of  demarca- 
tion, in  other  words,  between  the  human  bacillus  carrier  and  the 
patient  in  whom  a  focal  disease  is  chronic  for  long  periods  of  time  is 
not  sharply  circumscribed. 

7.  Contact  Infection. — The  direct  transmission  of  bacteria  from  man 
to  man  is  well  exemplified  in  the  venereal  diseases,  gonorrhea  and 
syphilis,  which  are  usually  transmitted  by  direct  contact.     Diseases 
of  the  respiratory  tract,  as  tuberculosis,  diphtheria  and  whooping- 
cough,  may  be  transmitted  directly  from  patient  to  patient  by  kissing, 
swapping  chewing  gum,  by  eating  utensils,  etc.     Soiled  fingers  may 
transmit  the  typhoid  bacillus  from  a  typhoid  patient  to  other  indi- 
viduals.    All  of  the   excrementitious    diseases  may  be  spread  in  a 
similar  manner,  under  certain  conditions. 

8.  Germinal  and  Prenatal  Infection. — True  germinal  infection  implies 
that  a  disease-producing  microorganism  is  carried  by  the  ovum  or 
spermatozoa  and  incorporated  in  the  embryo  prior  to  its  development. 
This  method  of  transmission  is  not  definitely  worked  out,  although  it 
has  been  claimed  that  syphilis  may  be  thus  transmitted  by  the  male 
to  the  ovum  in  utero,  the  mother  remaining  uninfected  by  the  disease. 

In  prenatal  infections  the  organisms  must  pass  the  placental 
barrier.  This  implies  that  the  fetus  becomes  infected  directly  from  the 
maternal  blood  stream,  or  by  continuity  of  growth  of  the  organisms 
through  the  placenta.  The  placental  form  of  infection  is  not  conceded 
by  all  observers,  but  it  is  reasonably  certain  that  congenital  syphilis 
may  be  contracted  thus.  Smallpox,  measles,  dysentery,  various 
pyogenic  infections,  and,  rarely,  pneumonia  are  occasionally  said  to  be 
prenatally  transmitted  to  the  fetus.  With  respect  to  tuberculosis, 
there  is  difference  of  opinion.  A  very  few  cases  are  on  record  in  which 
prenatal  infection  seems  almost  certainly  to  have  taken  place,  for  the 
newborn  infant  exhibited  lesions  which  were  so  far  advanced  that  no 
other  explanation  than  prenatal  infection  suffices  to  explain  them. 

C.  Portal  of  Entry;  Atria  of  Invasion. — The  bacteria  which  cause 
infection  in  the  human  body  may  be  provisionally  divided  into  two 
great  groups:  those  of  exogenous  origin,  which  are  not  habitual 
parasites  of  man;  and  those  of  endogenous  origin,  which  are  habitual 
parasites  of  man. 


PARASITIC  AND  PATHOGENIC  BACTERIA  97 

The  girat  majority  of  specific  microbic  diseases  (in  contradistinction 
to  non-specific  inflammations)  are  incited  by  bacteria  of  exogenous 
origin.  These  organisms  must  enter  the  host  directly  through  their 
respective  appropriate  atria  to  produce  characteristic  disease.  For 
example,  the  typhoid  bacillus  only  causes  typhoid  fever  when  the 
organism  is  swallowed  and  enters  the  body  through  the  intestinal 
tract.  Infection  of  a  skin- wound  with  typhoid  bacilli  will  not  result 
in  typhoid  fever.  Similarly,  cholera  vibrios  do  not  produce  the  disease 
cholera  unless  they  enter  the  body  through  the  gastro-intestinal  tract, 
although  if  cholera  vibrios  are  introduced  through  the  skin  in  experi- 
mental animals  they  tend  to  migrate  toward  the  intestinal  tract,  thus 
suggesting  a  special  affinity  for  the  intestinal  tissues.  Pathogenic 
bacteria  of  exogenous  origin  produce  in  general,  progressive  specific 
disease  from  man  to  man.  Bacteria  of  endogenous  origin,  on  the 
other  hand — those  which  occur  habitually  as  "  opportunists"  on  the 
surface  of  the  body  or  on  mucous  membranes  opening  to  the  exterior 
— ordinarily  exist  as  harmless  parasites.  They  may,  however,  and 
occasionally  do,  become  invasive,  inciting  local  or  generalized  inflam- 
matory reactions  as  a  rule,  rather  than  well-defined  clinical  syndromes 
which  are  frequently  so  characteristic  of  infections  with  exogenous 
pathogenic  bacteria.  The  bacteria  of  the  "opportunist"  type  do  not 
ordinarily  gain  entrance  to  the  tissues  of  the  body  through  sharply- 
circumscribed  atria  and  the  disease  they  produce  is  usually  not  epidemic 
in  character. 

1.  Skin  and  Adnexa — The  intact  skin  is  a  natural  barrier  which 
protects  the  underlying  tissues  of  the  body  from  bacterial  invasion. 
Its  free  exposure  to  the  environment  suggests  that  a  great  variety  of 
organisms  find  lodgment  upon  it  from  time  to  time;  a  majority  of 
these  organisms  are  harmless,  and  probably  transient  saprophytes 
which  come  and  go  irregularly.  The  moisture  and  excretions,  how- 
ever, appear  to  favor  the  limited  development  of  a  few  types  of  bac- 
teria, mainly  those  of  the  coccal  group,  which  occur  with  sufficient 
regularity  to  be  regarded  provisionally  as  habitually  parasitic  bacteria. 
Of  these,  the  pyogenic  cocci  are  usually  the  most  numerous;  they 
exist  as  "opportunists"  on  the  surface  of  the  skin  or  penetrate  into 
hair  follicles  and  the  ducts  of  the  cutaneous  glands,  ordinarily,  however, 
without  becoming  invasive  so  long  as  the  continuity  of  the  skin  is 
maintained.  Abrasions  and  cuts  furnish  a  portal  of  entry  to  the  sub- 
cutaneous tissues,  in  which  these  parasitic  bacteria  frequently  set  up 
inflammatory  reactions.  Friction  may  actually  force  them  through 


98         SAPROPHYTISM,  PARASITISM,  AND  PATHOGEN  ISM 

the  intact  skin.  The  plague  bacillus  and  certain  types  of  staphylococci 
are  said  to  pass  through  the  skin  occasionally  in  this  manner. 

Streptococci  and  staphylococci  are  the  more  common  habitual 
bacterial  parasites  found  on  the  skin.  Staphylococcus  epidermidis 
albus  (Welch),  a  variant  of  Staphylococcus  pyogenes  albus,  is  a  particu- 
larly common  factor  in  the  causation  of  the  troublesome,  but  relatively 
benign  stitch  abscesses  which  frequently  develop  where  sutures  are 
introduced  through  the  skin. 

The  damaged  skin  is  the  usual  portal  of  entry  for  spore-forming 
bacteria  as  well  as  the  cocci  mentioned  above.  Spores  of  the  bacilli 
of  tetanus,  anthrax,  symptomatic  anthrax,  malignant  edema  and  the 
"gas  bacillus,"  (B.  aerogenes  capsulatus,  Welch)  may  pass  to  the 
underlying  tissues  through  abrasions  of  the  skin  and  cause  either 
localized  infections  or  widely  distributed  lesions.  Even  so  insignifi- 
cant an  abrasion  as  an  insect  bite  may  furnish  the  necessary  atrium 
for  infection.  The  umbilicus  of  the  newborn  furnishes  a  portal  of 
entry  for  certain  bacteria;  particularly  severe  is  the  infection  of  the 
stump  of  the  umbilicus  with  B.  tetani,  causing  that  very  fatal  "tetanus 
neonatorum"  which  has  been  so  common  in  the  tropics  in  the  past. 
"Contused  wounds  and  compound  fractures  are  particularly  dangerous; 
the  inflamed  tissues  furnish  anaerobic  conditions  particularly  favoring 
the  growth  of  anaerobic  bacteria,  as  the  tetanus  and  gas  bacilli. 
Clean-cut  wounds  are  usually  less  liable  to  infection  with  anaerobic 
bacteria.  The  free  flow  of  blood  with  its  bactericidal  properties 
washes  out  many  bacteria,  inhibits  the  growth  of  residual  microbes, 
and  by  virtue  of  the  clot  which  soon  seals  the  wound  prevents  the 
entrance  of  other  organisms. 

The  sebaceous  secretions,  particularly  of  the  axilla  and  external 
genitalia,  are  good  culture-media  for  certain  acid-fast  bacteria,  par- 
ticularly B.  smegmatis.  The  cerumen  of  the  external  ear  is  frequently 
infected  with  Micrococcus  cereus  flavus,  and  the  puncture  of  the 
tympanic  membrane  may  lead  to  direct  infection  of  the  middle  ear 
from  the  outside,  with  this  or  other  organisms.  Infection  of  the 
middle  ear  may  also  take  place  directly  through  the  Eustachian  tube. 
The  blood  and  lymph  may  also  deposit  bacteria  in  the  middle  ear. 

The  conjunctiva,  by  virtue  of  its  very  exposed  position,  must 
receive  bacteria  upon  it  very  frequently.  Its  polished  surface  and 
the  mechanical  cleansing  by  the  flow  of  tears  (which  do  not  possess 
germicidal  properties)  usually  suffice  to  remove  adventitious  bacteria 
and  to  prevent  bacterial  development  under  ordinary  conditions.  The 


PARASITIC  AND  PATHOGENIC  BACTERIA  99 

conjunctival  sac,  which  receives  the  washings  from  the  conjunctiva, 
is  probably  the  recipient  of  many  bacteria;  of  these  B.  xerosis  occurs 
with  sufficient  regularity  in  the  conjunctival  sac  to  be  regarded  as  a 
normal  inhabitant.  The  pneumococcus  is  also  found  there.  These 
organisms  are  "opportunists,"  occasionally  causing  severe  acute 
conjunctivitis,  although  usually  they  are  benign.  Certain  bacteria 
affect  the  conjunctiva  fairly  readily.  Among  these  organisms,  the 
gonococcus  is  particularly  troublesome,  causing  a  most  severe  inflam- 
mation. Ophthalmia  neonatorum,  a  gonorrheal  infection  of  the  con- 
junctivse  of  the  newborn  of  infected  mothers,  has  been  in  the  past 
a  most  common  cause  of  blindness.  It  has  been  claimed  that  the 
meningococcus  may  occasionally  pass  from  the  eye  through  the  tear 
duct  to  the  nasal  cavity,  and  from  there  to  the  meninges. 

Subcutaneous  Tissue.- — Many  bacteria,  particularly  exogenous 
pathogenic  bacteria,  do  not  develop  in  the  subcutaneous  tissues,  as 
for  example,  the  majority  of  those  organisms  which  induce  specific  pro- 
gressive disease  from  man  to  man  such  as  typhoid  and  cholera  organ- 
isms. On  the  other  hand,  many  of  those  bacteria  which  are  habitually 
parasitic  on  the  skin  may  produce  infections  of  the  subcutaneous 
tissues  which  vary  in  severity  from  mild  inflammations  to  severe 
cellulitis.  The  staphylococci  and  streptococci  are  among  the  more 
important  of  this  type. 

Tonsils. — The  crypts  of  the  tonsils  afford  mechanical  protection 
to  bacteria  which  gain  access  to  them  and  the  secretions  and  tissue 
undoubtedly  provide  the  necessary  nutritive  elements,  consequently 
it  is  not  surprising  to  find  many  types  of  bacteria  in  them.  Staphy- 
lococci are  almost  invariably  present  and  streptococci,  particularly 
non-hemolytic  varieties,  are  very  common.  The  tonsils,  which  are 
in  very  direct  communication  with  the  lymphatic  system,  are  impor- 
tant atria  of  invasion,  particularly  for  streptococci,  and  many  cases 
of  low-grade  infections  of  the  body  appear  to  have  originated  from 
the  passage  of  bacteria  through  the  tonsils  to  the  tissues  of  the  body. 
The  extent  to  which  the  normal  tonsils  destroy  bacteria — their  value 
in  the  non-specific  initial  defense  of  the  body  against  bacterial  invasion 
in  other  words — is  not  clearly  established.  Generally  speaking,  how- 
ever, the  tonsils  appear  to  bear  the  brunt  of  attack  in  certain  diseases 
and  they  are  of  undoubted  importance  in  shielding  the  body  from 
invasion  through  the  lymphatic  tract  by  directly  holding  back  these 
bacteria.  The  promiscuous  removal  of  tonsils,  particularly  in  the 
young,  has  no  justification  from  available  knowledge.  The  removal 
of  diseased  tonsils  is  quite  a  different  matter. 


100       SAPROPHYTISM,  PARASITISM,  AND  PATHOGENISM 

Salivary  Glands. — The  salivary  glands  of  the  mouth  are  sometimes 
invaded  by  bacteria. 

Nasal  Cavity. — Large  numbers  of  bacteria,  indeed  practically  all 
known  bacteria  may  at  one  time  or  another  gain  access  to  the  nose 
through  the  inhalation  of  air  containing  dust,  by  droplet  infection, 
from  the  tear  ducts,  and  in  other  ways.  The  air  which  is  inhaled  is 
freed  from  bacteria  before  it  enters  the  trachea,  largely  during  its 
tortuous  passage  over  the  turbinates;  the  moist  surface  of  the  nasal 
mucosa  effectively  arrests  the  progress  of  bacteria,  which  adhere  to 
it.  The  constant  secretion  of  mucus  encloses  many  of  these  organ- 
isms, which  are  removed  mechanically  with  the  mucus.  There  is  no 
evidence  that  the  nasal  secretions  are  germicidal.  The  permanent 
nasal  flora  is  very  limited,  however.  The  pseudodiphtheria  bacillus 
is  very  frequently  found  there  and  pneumococci,  streptococci  and 
staphylococci  are  relatively  common.  The  true  diphtheria  bacillus 
is  found  in  the  nasal  cavity  of  about  1  per  cent,  of  healthy  individuals. 

Lungs. — The  expired  air  in  quiet,  normal  breathing  is  sterile: 
also,  the  inhaled  air  is  practically  sterile  before  it  reaches  the  bronchi, 
for  the  moist  tortuous  passages  of  the  nasal  cavity  mechanically 
retain  bacteria;  the  same  mechanism  prevents  the  expulsion  of  bac- 
teria during  exhalation,  unless  the  breath  is  expelled  forcibly  either 
through  the  nose  or  mouth.  Bacteria  leave  the  nose  or  mouth  in 
expired  air  only  when  the  expiration  is  forcible  enough  to  eject  finely 
divided  droplets  from  the  mouth  or  nose  respectively. 

The  lungs  are  protected  from  bacterial  invasion  not  only  by  the 
tortuous  nasal  air  passages,  but  by  the  ciliated  epithelium  which 
covers  the  surface  of  the  mucosa  of  the  bronchi  and  bronchioles. 
The  rhythmic  contractions  of  these  cilia  carry  upward  and  outward 
those  bacteria  which  may  have  penetrated  so  deeply  into  the  respira- 
tory passages.  Inhibition  of  the  activity  of  these  cilia  by  cold  or  other 
environmental  conditions  may  be  a  potent  factor  in  the  establishment 
of  infection  in  the  respiratory  tract.  Occasionally  bacteria  succeed 
in  reaching  the  terminal  bronchioles  and  alveoli  of  the  lungs :  they 
are  normally  removed  by  the  phagocytic  activity  of  leukocytes  (micro- 
phages)  or  of  certain  fixed  tissue  cells  (macrophages) .  In  spite  of 
these  barriers,  however,  the  lungs  occasionally  become  infected.  The 
pneumococcus  and  tubercle  bacillus  are  the  most  common  primary 
invaders  of  the  lungs.  Streptococci  are  more  frequently  secondary 
invaders,  although  many  primary  lobular  pneumonias  are  caused  by 
this  organism. 


PARASITIC  AND  PATHOGENIC  BACTERIA  101 

2.  Mucous  Membranes. — The  moist  surface  of  mucous  membranes 
makes  them  excellent  culture  media  for  many  bacteria  which  can 
grow  at  the  temperature  of  the  body.  The  physiological  secretions 
which  bathe  these  membranes,  with  the  exception  of  the  stomach, 
are  usually  without  germicidal  properties;  at  best,  their  antiseptic 
properties  are  wreak.  The  removal  of  bacteria  from  such  surfaces 
is  probably  for  the  most  part  mechanical.  The  secretion  of  mucus, 
which  has  been  shown  to  enclose  bacteria,  may  be  an  important  factor 
in  their  elimination. 

Mouth. — The  mouth  is  a  most  important  portal  of  entry  for  the 
great  majority  of  bacteria,  both  pathogenic  and  non-pathogenic, 
which  are  associated  with  man.  All  of  the  intestinal  bacteria,  harmful 
or  benign,  many  of  the  bacteria  which  are  associated  with  morbid 
processes  of  the  respiratory  tract,  and  several  which  induce  specific 
lesions  of  the  brain  and  spinal  cord  enter  through  this  atrium.  A 
great  majority  of  viruses  which  infect  the  respiratory  tract  and  the 
cerebrospirial  axis  also  leave  the  body  through  the  mouth  or  nose. 

The  normal  flora  of  the  mouth  is  quite  varied,1  including  not  only 
bacteria  which  are  ordinarily  regarded  as  harmless,  but  also  organisms 
which  occasionally  or  frequently  incite  disease.  Thus,  from  20  to  40 
per  cent,  of  healthy  individuals  living  in  large  cities  harbor  typical 
and  apparently  virulent  pneumococci  in  their  mouths;2  about  2  per 
cent,  of  school  children  harbor  typical  diphtheria  bacilli  in  their 
mouths.3  Rarely,  tubercle  bacilli  have  been  detected  in  the  mouths 
of  apparently  normal  individuals. 

It  is  worthy  of  note  that  an  occasional  abscess  in  the  cervical  region 
may  contain  spiral  organisms;  frequently  a  careful  examination  will 
reveal  a  sinus  connecting  the  abscess  with  the  mouth,  perhaps  origi- 
nating at  the  base  of  a  carious  tooth.  Dental  caries  is  usually  regarded 
as  a  bacteriological  process.  The  removal  of  bacteria  from  the  teeth 
and  gums  can  not  be  satisfactorily  accomplished  by  antiseptic  mouth 
washes  and  the  saliva  possesses  no  germicidal  properties.  Bacteria 
are  removed  from  the  teeth  mechanically  by  friction  and  are  trans- 
ported from  the  mouth  to  the  stomach  during  the  processes  of  mas- 
tication and  deglutition.  '  The  oral  flora  is  most  numerous  before 

1  For  full  literature  and  descriptions  see  Miller,  Die  Mikroorganismen  der  Mundhohle, 
Leipzig,  1892,  and  Goadby,  Mycology  of  the  Mouth,  1903. 

2  Recent  observations  by  Cole  and  his  associates  indicate  that  the  ordinary  "mouth" 
pneumococci  differ  in  their  serological  reactions  from  pneumococci  isolated  directly 
from  pneumonia  lesions. 

3  Moss,  Guthrie  and  Gelien  have  found  a  much  larger  proportion  of  diphtheria  bacillus 
carriers  during  a  period  when  diphtheria  was  epidemic. 


102    , •' SAPRaP-HYTISM,  PARASITISM,  AND  PATHOGENISM 

eating  and  almost  absent  immediately  after  eating  a  hearty  meal. 
Tubercle  bacilli  are  swallowed  thus  and  many  of  them  eventually 
appear  in  the  feces. 

Stomach. — The  acidity  of  the  stomach  during  gastric  digestion, 
by  virtue  of  the  free  hydrochloric  acid  of  the  gastric  juice,  is  a  potent 
factor  in  the  destruction  of  bacteria  which  reach  the  stomach  both 
from  the  mouth  and  the  respiratory  tract.  Mineral  acids  are  much 
more  powerful  germicides  than  organic  acids.  The  normal  stomach, 
therefore,  is  quite  free  from  inflammations  or  irritations  attributable 
to  the  activity  of  bacteria.  Many  bacteria,  however,  run  the  gauntlet 
of  the  stomach  successfully,  especially  when  the  stomach  is  empty 
(when  the  concentration  of  hydrochloric  acid  is  very  low)  and  pass 
into  the  intestinal  tract,  where  the  conditions  are  much  more  favorable 
for  their  growth.  The  passage  of  bacteria  through  the  stomach  prob- 
ably takes  place  either  very  early  in  gastric  digestion,  when  the 
hydrochloric  acid  is  not  at  its  "digestive  concentration"  (about  0.2 
per  cent.),  or  after  gastric  digestion  has  ceased.  When  water  or 
other  fluids  are  drunk,  which  do  not  call  forth  gastric  juice,  bacteria 
doubtless  pass  through  the  stomach  unharmed,  and  it  is  probable 
that  organisms  included  mechanically  within  food  particles  may 
occasionally  escape  the  action  of  the  gastric  acidity. 

Certain  aciduric  bacteria1  and  even  yeasts  which  are  tolerant  of 
acid  may  be  found  occasionally  in  the  normal  stomach,  but  rarely 
or  never  pathogenic  bacteria.  Abnormally,  particularly  when  the 
hydrochloric  acid  is  deficient,  many  bacteria  are  found  in  the  stomach 
contents.  Obstruction  of  the  pylorus  tends  to  increase  the  number 
of  bacteria  in  the  stomach  by  promoting  stasis  of  food.  This  con- 
dition is  particularly  common  in  carcinoma  of  the  pylorus.  The 
Oppler-Boas  bacillus,  sometimes  called  B.  geniculatus,  one  of  the 
aciduric  bacteria,  is  so  frequently  found  in  this  pathological  condition 
it  was  at  one  time  supposed  to  be  an  accessory  factor;  it  is  now  known 
to  have  no  relationship  to  gastric  carcinoma.  B.  geniculatus  is  also 
found  very  commonly  in  cases  of  achlorhydria.  Sarcina  ventriculi 
is  also  found  in  similar  conditions. 

The  gastric  acidity  will  destroy  the  toxins  of  B.  diphtherise  and 
B.  tetani;  the  toxin  of  B.  botulinus  is  not  inactivated  by  the  gastric 
juice.  The  toxins  of  the  paratyphoid  group  of  bacteria  also  appear 
to  be  resistant  to  gastric  digestion. 

1  Kendall,  Jour.  Med.  Research,  1910,  N.  S.,  xviii,  153. 


PARASITIC  AND  PATHOGENIC  BACTERIA  103 

Intestines. — The  abundant  intestinal  contents,  which  vary  some- 
what in  composition  and  reaction  at  different  levels,  provide  conditions 
which  make  the  intestinal  tract  a  very  efficient  combined  incubator 
and  culture  medium.  Many  kinds  of  bacteria  may  theoretically  find 
conditions  well  adapted  to  their  rapid  development  there  and  it  is 
not  surprising  to  find  that  bacterial  proliferation  is  greater  both  in 
nature  and  extent  in  the  intestinal  tract  than  in  any  other  known 
medium.  It  has  been  conservatively  estimated  that  the  average 
daily  fecal  excretion  of  bacteria  in  a  healthy  adult  on  a  normal  diet 
is  expressed  by  the  truly  enormous  number,  33  x  1012.  About  47  per 
cent,  of  the  nitrogen  of  the  feces  is  contained  in  the  bodies  of  these 
bacteria  which,  when  dried,  weigh  nearly  0.5  gram. 

The  upper  level  of  the  intestinal  tract,  particularly  the  duodenum, 
is  relatively  free  from  bacteria  during  interdigestive  periods.  The 
duodenal  bacterial  population  increases  rapidly  when  food  enters 
this  section  of  the  alimentary  canal  and  decreases  when  the  food 
passes  to  lower  levels.  The  numbers  of  bacteria  increase  very  greatly 
where  stasis  of  food  becomes  more  marked  and  in  the  cecum  and 
large  intestines  generally  there  are  continually  present  enormous 
numbers  of  bacteria.1 

The  types  of  bacteria  found  in  the  intestinal  tract  are  influenced 
markedly  by  the  nature  of  the  food  of  the  host  and  by  the  ability  of 
the  organisms  themselves  to  change  their  metabolism  to  meet  varia- 
tions in  the  composition  of  this  food.  Those  bacteria  which  can  best 
meet  alternations  in  diet  of  the  host  are  the  ones  which  naturally 
persist.  The  bacteria  contained  in  the  food  itself  may  also  play  a 
prominent  part  in  determining  the  nature  of  the  organisms  which 
are  found  in  the  intestinal  tract.  The  colon  bacillus  is  particularly 
labile  in  meeting  dietary  alternations  in  the  intestines  and  this  organism 
constitutes  about  80  per  cent,  of  the  bacteria  which  can  be  isolated 
from  the  feces  of  the  adult. 

At  birth  the  intestinal  tract  is  sterile  and  the  embryonal  feces,  the 
meconium,  which  is  passed  during  the  first  eighteen  hours  after  birth, 
is  sterile.  Following  this  period  of  sterility  there  is  a  period  lasting 
about  three  days  on  the  average,  in  which  various  adventitious  organ- 
isms are  met  with  in  the  dejecta.  The  normal  nursling  flora  begins 
to  appear  by  the  end  of  the  third  day,  following  the  ingestion  of 
breast  milk.  The  dominant  organism  of  this  nursling  flora  is  ordi- 
narially  an  obligate  anaerobe,  Bacillus  bifidus,  which  is  one  of  the 

1  Kendall,  Jour.  Med.  Research,  1911,  xxv,  126-130. 


104       SAPROPHYTISM,  PARASITISM,  AND  PATHOGENISM 

best  known  examples  of  obligately  fermentative  organisms.  It  does 
not  thrive  on  a  purely  protein  diet  but  requires  carbohydrate,  which 
is  normally  supplied  by  the  breast  milk.  Breast  milk,  it  will  be  remem- 
bered, contains  on  the  average  about  7  per  cent,  of  lactose,  3  per  cent, 
fat  and  but  1.5  per  cent,  protein.  The  proportion  of  carbohydrates 
to  protein  in  the  diet  decreases  as  the  infant  becomes  older  and  the 
diet  becomes  more  liberal,  and  this  decrease  in  the  percentage  of 
carbohydrate  is  associated  with  a  diminution  in  the  number  of  the 
obligately  fermentative  bacteria,  particularly  of  Bacillus  bifidus,  and 
their  gradual  replacement  by  organisms  which  can  thrive  well  on  a 
diet  containing  variable  proportions  of  carbohydrate  and  protein.1 

Bacillus  coli  is  a  most  labile  organism  with  respect  to  its  ability 
to  develop  in  the  carbohydrate  and  protein  constituents  of  the  intes- 
tinal contents  at  the  ileocecal  region  and  lower  levels;  this  organism 
is  represented  to  the  extent  of  fully  80  per  cent,  in  the  feces  of  healthy 
men.  Smaller  numbers  of  other  bacteria,  as  Micrococcus  ovalis, 
Bacillus  acidophilus,  B.  proteus,  B.  mesentericus,  B.  aerogenes  cap- 
sulatus  and  many  other  varieties  are  found  transiently  or  semi-per- 
manently  in  the  intestinal  contents.  Exogenic  bacteria  occasionally 
invade  the  tissues  of  the  body  through  the  intestinal  mucosa.  Thus 
typhoid,  paratyphoid  and  dysentery  bacilli  and  cholera  vibrios  may 
produce  severe  infections.  The  tubercle  bacillus  may  pass  through 
the  apparently  intact  intestinal  wall  without  leaving  any  evidence 
of  its  passage.  It  is  supposed  that  this  organism  penetrates  the 
intact  mucosa  and  enters  lymphatic  channels  suspended  in  fats  and 
eventually  proliferates  in  deeper  tissues. 

3.  Genito-Urinary  System. — Vagina. — The  vagina  has  an  acid  reac- 
tion and  it  harbors  very  few  bacteria,  but  immediately  afterchild- 
birth  the  reaction  may  become  temporarily  alkaline.  The  bacillus 
of  Doderlein,  however,  occurs  so  commonly,  that  it  may  be  provision- 
ally regarded  as  a  normal  inhabitant  and  a  few  strains  of  aciduric 
cocci  are  not  infrequently  detected  in  cultures  from  the  fundus  of  the 
vagina.  The  Gonococcus  and  Treponema  pallidum  are  the  more 
common  pathogenic  organisms  whose  portal  of  entry  is  the  vagina. 

Uterus. — The  normal  uterus  is  sterile  and  the  acid  reaction  of  the 
vagina  and  the  closure  of  the  cervix  uteri  tends  to  maintain  sterility 
under  normal  conditions.  During  menstruation  and  childbirth  the 
mechanical  defenses  of  the  uterus  are  impaired.  The  organ  itself 
appears  to  possess  no  specialized  powers  of  resistance  to  infection. 

1  A  more  detailed  discussion  on  intestinal  bacteria  and  their  significance  will  be  found 
in  Chapter  xxx. 


PARASITIC  AND  PATHOGENIC  BACTERIA  105 

Urethra. — The  urethra  in  health  is  practically  free  from  bacteria. 
The  flow  of  urine  mechanically  frees  it  from  bacteria.  The  external 
orifice  of  the  urethra,  however,  frequently  contains  an  acid-fast  organ- 
ism, Bacillus  smegmatis,  which  can  be  differentiated  from  the  tubercle 
bacilli  only  by  animal  inoculation,  and,  very  frequently,  Bacillus  coli. 
The  gonococcus  and  Treponema  pallidum  may  invade  the  tissues 
through  the  urethra. 

Urinary  Bladder  and  Ureter. — The  slightly  alkaline  reaction  of  the 
urine  affords  a  good  culture  medium  for  many  bacteria  and  infection 
of  the  bladder  by  B.  coli,  B.  proteus,  B.  typhosus  and  other  micro- 
organisms is  by  no  means  uncommon.  It  is  probable  that  infection 
occurs  much  more  frequently  through  the  blood  or  lymph  than  through 
an  ascending  infection  from  the  urethra.  B.  proteus  appears  to  grow 
with  great  luxuriance  in  the  urinary  bladder  and  a  typical  cystitis 
may  be  readily  incited  in  dogs  by  injecting  virulent  cultures  of  the 
organism  directly  into  the  bladder.  Occasionally  a  descending  infec- 
tion from  an  inflamed  kidney  may  result  in  cystitis:  whether  a  true 
ascending  infection  through  the  ureter  to  the  kidney  takes  place  is 
not  definitely  proven. 

Kidneys. — The  kidneys  are  normally  free  from  bacteria,  but  infec- 
tion of  one  or  both  kidneys  through  the  blood  stream  is  a  well-estab- 
lished phenomenon.  A  variety  of  organisms  may  thus  infect  the 
kidney.  The  cocci  of  suppuration  frequently  incite  acute  nephritis 
and  tubercle  bacilli  induce  chronic  infection.  Theoretically,  any 
invasive  organism  which  enters  the  blood  stream  may  localize  in  the 
kidney  and  establish  metastatic  foci  there.  The  organ  is  susceptible 
to  specific  bacterial  toxins  as  well  as  to  the  bacteria  themselves. 

I).  Where  Bacteria  Multiply  in  the  Body. — Practically  no  organ 
or  part  of  the  body,  except  such  structures  as  the  nails,  are  free  from 
invasion  with  one  or  another  kind  of  organism.  The  obvious  com- 
plexity of  the  subject  makes  it  difficult  or  even  impossible  to  present 
in  concrete  form,  a  statement  which  shall  indicate  specifically  the 
types  of  organisms  which  incite  infection  in  association  with  the 
particular  organs  or  tissues  where  they  become  localized.  It  is  impor- 
tant in  this  connection,  however,  to  remember  that  a  great  majority 
of  progressively  pathogenic  bacteria  exhibit  rather  marked  affinities 
for  special  tissues,  and  that  they  invade  the  tissues  through  definite 
atria.  The  organisms  which  are  habitually  parasitic,  on  the  contrary 
—the  "opportunists" — as  Theobald  Smith  has  so  clearly  pointed  out, 
are  less  exacting  in  this  respect,  as  a  rule,  and  they  may  invade  the 


106       SAPROPHYTISM,   PARASITISM,  AND  PATHOGENISM 

tissues  whenever  the  natural  barriers — skin,  mucous  membranes,  and 
so  on — weaken  and  become  vulnerable. 

The  following  table  indicates  the  more  common  and  important 
bacteria,  parasitic  or  pathogenic,  which  may  invade  the  tissues,  and 
the  organs  where  they  tend  to  localize  and  develop. 

SKIN: 

Staphylococcus  and  streptococcus  groups. 

Acid-fast  group:   tubercle  bacilli,  lepra  bacilli,  smegma  bacilli. 

Anaerobic  group:   tetanus,  gas  bacillus. 

Anthrax. 

"Bottle"  bacillus  (spore  of  Melassez). 
NOSE,  THROAT  AND  ADNEXA: 

Staphylococcus  group. 

Streptococcus  and  pneumococcus  group. 

Diphtheria  and  pseudodiphtheria  group. 

Influenza  and  pertussis  group. 

Pneumobacillus,  rhinoscleroma  and  ozena  group. 

Bacillus  fusiformis  and  spirillum  group. 

Meningococcus  and  catarrhalis  group. 

Acid-fast  group — 'Chiefly  tubercle  bacilli  and  leprosy. 

Blastomycetes  and  hyphomycetes. 

Virus  of  poliomyelitis  and  unknown  viruses,  mumps,  etc. 

(Organisms  of  dental  caries  and  pyorrhea  not  included  above.) 
EYE  AND  EAR: 

Streptococcus  and  pneumococcus  group. 

Staphylococcus  group. 

Diphtheria  and  pseudodiphtheria  group. 

Influenza  group. 

Koch- Weeks  and  Morax-Axenfeld  group. 

Gonococcus. 

Proteus  group. 

Pyocyaneus  group. 
LUNGS  : 

Streptococcus  and  pneumococcus  group. 

Pneumobacillus  group. 

Acid-fast  group:   tubercle  bacillus. 

Influenza  and  pertussis  group. 

Plague  bacillus,  anthrax  bacillus  and  B.  psittacosis. 

Colon  and  typhoid  group. 

Actinomyces  and  hyphomycetes. 
PELVIC  ORGANS: 

Streptococcus  and  Staphylococcus  group. 

Gonococcus  and  Treponema  pallidum. 

Tubercle  bacillus  and  smegma  bacillus. 

Micrococcus  melitensis. 
SEROUS  FLUIDS: 

1.  Cerebrospinal  fluid: 

(a)  Fluid  usually  clear:  tubercle  bacillus  and  Treponema  pallidum.  Virus 
of  poliomyelitis. 

(&)  Fluid  turbid:  Pneumococcus,  streptococcus,  meningococcus,  B.  influ- 
enza, B.  typhosus,  B.  coli. 

2.  Pleural  and  pericardial  fluids: 

(a)  Fluid  usually  clear:    tubercle  bacillus. 

(&)  Fluid  turbid  as  a  rule:  pneumococcus,  streptococcus,  B.  influenzce, 
Pneumobacillus  group,  Bacillus  typhosus,  Staphylococcus. 

3.  Peritoneal  fluid: 

Streptococcus  group. 
Coli  and  typhoid  group. 
Tubercle  bacillus  (?). 


BALANCED  PATHOGENISM;  EPIDEMIOLOGY  107 

BLOOD: 

Streptococcus  and  pneumococcus  group. 

Staphylococcus  group. 

Typhoid,  paratyphoid  and  dysentery  group. 

B.  coli. 

Recurrent  fever  and  treponemata. 

B.  pestis. 

Certain  filterable  viruses:    Yellow  fever,  poliomyelitis  (?). 

Tubercle  bacillus  (occasionally). 
INTESTINAL  CONTENTS,  FECES: 

B.  bifidus  and  B.  acidophilus  group  (chiefly  in  infants). 

B.  coli,  B.  lactis  aerogenes,  proteus  and  cloacae  group. 

Alcaligenes,  paratyphoid,  typhoid  and  dysentery  group. 

Streptococcus  and  Micrococcus  ovalis  groups. 

Mucosus  capsulatus  group. 

Spore-forming  group:  Aerobic — B.  mesentericus,  B.  subtilis,  B.  anthracis. 

Anaerobic — B.  aerogenes  capsulatus,  B.  tetani,  B.  botulinus. 

Acid-fast  group:    tubercle  bacilli,  bovine  and  human;   grass  bacilli. 

Spiral  group:   Vibrio  cholerse,  Sp.  of  Finkler  and  Prior. 

E.  Where  and  How  Bacteria  Escape  from  the  Body. — It  appears 
from  foregoing  considerations  that  those  microorganisms  which  are 
progressively  pathogenic  for  man  habitually  invade  the  tissues  through 
atria  characteristic  for  each  microbe.  Their  escape  from  the  tissues 
through  appropriate  channels  in  direct  communication  with  the  out- 
side is  equally  important.  Bacteria  of  the  "opportunist"  type  fre- 
quently perish  within  the  tissues  because  they  lack  a  perfected 
mechanism  of  escape  to  the  outside.  Progressively  pathogenic  bac- 
teria leave  the  body  through  two  principal  avenues — the  mouth  and 
nose,  and  the  feces.  Less  commonly,  certain  types  may  pass  to  the 
outside  in  the  urine.  The  skin  is  not  a  very  important  factor  in  the 
elimination  of  pathogenic  bacteria.  The  paths  of  pathogenic  bacteria 
from  the  tissues  to  the  outside  are  varied,  but  very  constant  for  each 
special  organism  and  the  discussion  of  this  phase  of  their  activity  is 
reserved  for  the  Section  on  Specific  Organisms. 

VI.     BALANCED   PATHOGENISM;  EPIDEMIOLOGY. 

It  has  been  helpful,  for  clearness  and  discussion,  to  distinguish 
rather  sharply  Setween  parasitic  and  pathogenic  bacteria  and  in  a 
majority  of  specific  instances  such  a  differentiation  can  be  readily 
established.  There  is  no  hard  and  fast  line  of  demarcation,  however, 
between  organisms  of  the  "opportunist"  type  and  those  progressively 
pathogenic,  for  it  is  undoubtedly  true  that  some  "opportunists"  may 
exhibit  epidemic  tendencies  for  limited  periods  if  a  combination  of 
conditions  arise  which  favor  the  distribution  of  the  organisms  and 
either  increase  the  invasive  powers  of  the  microbe  or  decrease  the 
resistance  of  the  host.  The  limited  spread  of  such  bacteria  is  far  more 


108       SAPROPHYTISM,  PARASITISM,  AND  PATPIOGENISM 

frequently  attributable  to  unusually  direct  transfer  of  organisms  by 
a  common  vehicle  through  a  series  of  susceptible  hosts  than  to  the 
escape  of  the  microbes  from  one  host  to  another.  Thus,  milk-borne 
epidemics  of  septic  sore  throat  may  be  extensive  and  involve  many 
patients,  but  secondary  transfer  from  man  to  man  is  relatively  uncom- 
mon. These  bacteria  have  not,  as  a  rule,  perfected  their  mechanism 
of  escape  from  the  tissues  of  one  host  to  those  of  another.  The 
epidemics  are  usually  of  brief  duration  and  it  is  probable  that  the 
surviving  microbes  return  to  their  original  parasitic  state. 

Of  far  greater  importance  is  a  probable  tendency  of  many  progres- 
sively pathogenic  bacteria  to  act  more  and  more  on  the  defensive; 
to  gradually  disembarrass  themselves,  on  the  one  hand,  of  the  offen- 
sive weapons  which  originally  conferred  upon  their  possessors  the 
ability  to  invade  their  host,  and,  on  the  other  hand,  to  perfect  what- 
ever defensive  weapons  they  may  have  possessed  the  rudiments  of.1 
Such  a  change,  as  Theobald  Smith  has  pointed  out,  would  be  difficult 
to  detect,  because  an  elimination  of  the  more  aggressive  type  and  its 
gradual  replacement  by  a  strain  in  which  the  defensive  elements  were 
more  prominently  represented  would  require  years  for  its  accomplish- 
ment. Such  a  change  in  the  activities  of  the  microorganisms  would 
probably  be  accompanied  by  reciprocal  activities  of  the  host,  so  that 
eventually  a  strain  of  microorganisms  would  be  evolved  which  had 
reached  a  state  of  relative  equilibrium  with  the  host.  Unusually 
virulent  strains  of  microbes  would  tend  to  perish  with  their  hosts, 
and  unusually  susceptible  hosts  would  tend  to  perish  with  their 
invaders.  A  mutual  adjustment  of  virulence  and  resistance  between 
the  surviving  hosts  and  microbes  would  lead  eventually  to  one  of 
three  conditions: 

1.  Gradual  extinction  of  the  microorganism; 

2.  The  gradual  assumption^  of  a  parasitic  or  "  opportunist "  exist- 
ence, or 

3.  A  more  perfect  pathogenism  in  which  the  mechanism  of  invasion, 
multiplication  within  the  tissues  and  escape  to  other  hosts  is  accom- 
plished without  acute  damage  to  the  host. 

It  might  well  happen  that  the  introduction  of  such  "balanced" 
strains  into  new  fields  would  lead  to  temporary  disaster,  as  for 
example,  the  highly  fatal  epidemic  of  measles  when  this  virus  first 
gained  a  foothold  in  the  South  Sea  Islands. 

1  Theobald  Smith  (Some  Problems  in  the  Life  History  of  Pathogenic  Microorganisms, 
Am.  Med.,  1904,  viii,  711)  clearly  stated  and  discussed  this  hypothesis  over  a  decade 
ago,  and  it  is  surprising  how  little  cognizance  has  been  taken  of  it. 


BALANCED  PATHOGENISM;  EPIDEMIOLOGY  109 

Theobald  Smith1  has  mentioned  the  diphtheria  bacillus  as  an 
organism  which  possibly  exhibits  a  tendency  toward  a  parasitic  exis- 
tence. The  toxin  of  the  diphtheria  bacillus  is  not  a  poison  specific 
for  man;  many  animals,  as  the  horse  and  guinea-pig,  are  very  sus- 
ceptible to  it.  Yet  the  diphtheria  bacillus  is  almost  obligately  a  human 
pathogen.  The  ever-increasing  occurrence  of  avirulent,  non-toxin 
producing  strains  which  are  otherwise  perfectly  typical,  and  the 
frequent  occurrence  of  individuals  whose  serum  contains  small 
amounts  of  natural  antitoxin  might  be  interpreted  as  an  indication 
that  strains  of  this  organism  are  becoming  gradually  accustomed  to 
a  purely  parasitic  existence  in  the  upper  respiratory  tract  of  man  on 
the  one  hand,  and  that  man  has  acquired  some  specific  resistance  to 
the  microbe  on  the  other  hand. 

The  tubercle  bacillus  (typus  humanus)  is  an  excellent  example  of 
an  exquisitely  balanced  pathogenic  microorganism.  Its  metabolism  is 
not  markedly  different  from  that  of  the  host  and  the  typical  disease 
excited  by  it  is  focal,  chronic,  and  slow-going.  Years  may  elapse  before 
the  host  finally  succumbs.  The  development  of  the  organisms  within 
the  tissues  of  the  host  does  not  appear  to  lead  to  the  formation  of 
substances  which  arouse  the  latent  offensive  and  defensive  mechanism 
of  the  host  to  acute  antagonism.  During  this  long  period  the  tubercle 
bacilli  establish  communication  with  the  outside  and,  in  a  majority 
of  cases,  countless  myriads  of  bacilli  escape  from  the  host  before 
death  removes  him  as  a  source  of  infection.  Occasionally  tubercle 
bacilli  become  widely  disseminated  in  the  body,  causing  rapidly  fatal, 
generalized  miliary  tuberculosis.  These  organisms  perish  with  their 
host. 

It  is  well  known  that  the  virulence  of  bacteria,  many  kinds  at  least, 
can  be  increased  decidedly  by  passage  from  animal  to  animal  by 
providing  an  artificial  portal  of  entry  and  of  exit  from  animal  to 
animal.  This  is  accomplished  by  injecting  the  organisms  into  a 
first  animal  and  reinjecting  them,  at  brief  intervals,  into  other  animals. 
In  such  instances  there  is  a  direct  continuity  of  growth  from  animal 
to  animal,  greater  than  is  met  with  in  naturally  occurring  infections. 
It  is  worthy  of  note  that  bacteria  of  the  "opportunist"  type  are,  gen- 
erally speaking,  more  successfully  exalted  in  virulence  under  these 
conditions  than  the  progressively  pathogenic  forms. 

There  is  yet  another  feature  of  Pathogenism  which  is  worthy  of 
note.  From  time  to  time  almost  any  bacterial  disease,  for  example, 

1  Theobald  Smith,  loc.  cit. 


110       SAPROPHYTISM,  PARASITISM,  AND  PATHOGENISM 

typhoid,  plague  or  influenza,  may  leap  suddenly  to  epidemic  propor- 
tions, spread  rapidly  and  then  subside  again,  to  be  succeeded  by 
sporadic  cases  which  gradually  diminish  in  numbers  and  in  severity. 
The  bacteria  causing  these  outbreaks  appear  to  acquire  somehow  and 
somewhere,  an  unusual  degree  of  invasiveness  and  they  spread  rapidly, 
especially  in  thickly  settled  areas,  and  as  rapidly  lose  their  unusual 
activities  and  subside  to  what  appears  to  be  their  usual  level  of  viru- 
lence. It  is  very  probable  that  those  strains  of  pathogenic  bacteria 
in  general,  which  suddenly  acquire  unusual  virulence  are  short-lived, 
partly  because  their  hosts  perish  before  the  microbes  can  escape  to 
new  hosts.  Not  infrequently,  these  or  similar  epidemics  are  preceded 
by  mild,  atypical  disease,  which  may  not  be  specifically  recognized, 
and  during  this  initial  period  the  bacteria  may  be  quite  widely  dis- 
tributed.1 

1  Kendall,  Boston  Med.  and  Surg.  Jour.,  1915,  clxxii,  851. 


CHAPTER  VI. 
IMMUNITY  AND  INFECTION. 


2.  Passive  Immunity. 

(a)  Antibody      Im- 
munity. 
(6)  Chemotherapy. 

3.  Mixed,    Active    and 

Passive  Immunity. 
II.  INFECTION — PRIMARY  AND  SEC- 
munity.  .  ONDARY. 

1.  Active  Immunity.  A.  Defenses  of  the  Host,  Non- 

(a)  Natural     Ac-  specific  and  Specific. 


GENERAL  PHENOMENA  OF  IMMUNITY. 
I.  CLASSIFICATION  OF  IMMUNITY. 

A.  Natural  or   Inherited   Im- 

munity. 

1.  Racial. 

2.  Individual. 

B.  Acquired   or   Induced    Im- 


quired    Im- 
munity. 
(6)  Artificial       Ac- 


Ill.  THEORIES  OF  IMMUNITY. 

A.  The  Humoral  Side-chain  or 
Ehrlich  Theory   of    Im- 


quired       Im-  munity. 


munity. 


B.  The  MetchnikorT  or  Phago- 
cytic  Theory  of  Im- 
munity. 


GENERAL   PHENOMENA   OF   IMMUNITY. 

IT  has  long  been  recognized  that  man  and  animals  exhibit  refrac- 
toriness to  infection  with  specific  bacteria  or  other  microorganisms 
which  cause  serious  epizootics  in  closely  related  animals.  Man  is, 
as  a  rule,  quite  free  from  the  epizootic  diseases  of  animals  domesticated 
by  him,  and  the  domestic  animals  are  usually  not  infected  with  the 
organisms  which  incite  progressive  disease  in  man.  Thus,  man  does 
not  contract  chicken  cholera  and  domestic  animals  do  not  become 
infected  with  the  typhoid  bacillus.  Furthermore,  closely  related 
animal  species  may  exhibit  striking  differences  in  susceptibility  to 
the  same  disease;  for  example,  field  mice  are  readily  infected  with 
the  glanders  bacillus,  but  house  mice  are  quite  resistant  to  infection 
with  this  organism,  and  ordinary  sheep  readily  succumb  to  anthrax 
although  Algerian  sheep  are  practically  immune  to  infection  with  the 
anthrax  bacillus. 

This  inherent  or  congenital  resistance  or  refractoriness  to  infection 
with  a  specific  microorganism,  when  general  among  the  individuals  of 
a  species  or  group  of  animals  or  of  man  is  termed  natural  or  inherited 
immunity.  It  is  not  necessarily  absolute;  lowering  the  natural  resis- 
tance of  the  individual  may  render  him  susceptible  to  infection.  Thus 


112  IMMUNITY  AND  INFECTION 

hunger,  experimental  (phloridzin)  diabetes,  fatigue  produced  by  pro- 
longed exercise  in  treadmills,  and  excessive  chilling  by  the  removal 
of  hair  have  been  shown  to  decrease  resistance  in  experimental  animals. 

It  is  also  a  matter  of  common  observation  that  the  uniform  exposure 
of  a  number  of  theoretically  susceptible  individuals  of  the  same  species 
to  a  virus  does  not  lead  to  uniform  infection;  a  certain  small  number 
usually  perish,  a  larger  proportion  become  mildly  or  severely  ill  and 
recover.  The  greatest  number  are  not  especially  affected,  as  a  rule. 
Those  individuals  who  escape  infection  in  one  epidemic  may  succumb 
to  infection  during  a  subsequent  epidemic  of  the  same  disease.  This 
phenomenon  of  individual  variation  in  susceptibility  is  well  exempli- 
fied in  water-  and  milk-borne  epidemics  of  typhoid  fever  where  typhoid 
bacilli  are  widely  distributed  in  a  water  or  milk  supply.  A  small 
number  become  infected,  but  the  greater  number  escape  the  disease. 
The  incidence  of  scarlet  fever,  of  diphtheria  or  of  other  infectious 
diseases  among  the  members  of  the  same  family  frequently  illustrates 
this  same  phenomenon.  This  resistance  to  infection  exhibited  by 
certain  individuals  of  a  susceptible  species  is  termed  inherited 
immunity. 

Susceptible  individuals  who  survive  a  naturally  acquired  or  arti- 
ficially induced  infection — as  smallpox,  measles,  typhoid  fever  or 
vaccinia — are  frequently  resistant  or  refractory  to  subsequent  infec- 
tion with  the  same  virus.  They  have  developed  a  resistance  to  specific 
infection,  they  have  acquired  immunity,  in  other  words.  This  type 
of  immunity,  which  results  from  actual  infection,  is  termed  active, 
acquired  immunity.  It  is  the  outcome  of  a  successful  struggle  between 
the  host  and  the  invading  microbe  during  which  the  former,  through 
cellular  activity,  produces  or  increases  antibodies  specifically  inimical 
to  the  latter.  The  immunity  which  is  thus  laboriously  produced 
is  frequently  fairly  persistent.  It  is  more  commonly  observed  fol- 
lowing invasion  by  exogenous,  progressively  pathogenic  bacteria  than 
infection  with  endogenous  microorganisms  of  the  "opportunist"  type. 
Indeed,  infection  with  the  latter  not  infrequently  results  in  increased 
susceptibility  to  subsequent  infection  with  the  same  species  of  microbe. 
Thus,  recovery  from  one  attack  of  typhoid  fever  usually  confers  last- 
ing immunity  upon  the  individual;  one  attack  of  lobar  pneumonia, 
on  the  other  hand,  appears  to  predispose  the  individual  to  subsequent 
infection  with  the  pneumococcus. 

The  injection  of  specific  immune  substances  or  antibodies  into 
susceptible  individuals  may  confer  upon  them  transient  or  temporary 


CLASSIFICATION  OF  IMMUNITY  113 

immunity  to  the  specific  infection;  the  host  is  a  passive  recipient  of 
antibodies  in  such  instances.  These  alien  antibodies,  however,  soon 
diminish  in  potency  or  disappear,  leaving  the  susceptibility  of  the  indi- 
vidual to  infection  at  its  original  level.  Immunity  induced  by  the 
injection  of  specific  antibodies  is  termed  passive  acquired  immunity. 
The  transitory  immunity  to  diphtheria  or  tetanus  following  the 
injection  of  diphtheria  or  tetanus  antitoxin  is  an  example  of  passive 
acquired  immunity. 

Immunity  may  be  localized  or  general  in  the  same  individual,  and 
different  individuals  frequently  exhibit  varying  degrees  of  resistance 
or  susceptibility  to  the  same  virus. 

I.     CLASSIFICATION   OF  IMMUNITY. 

Both  immunity  and  susceptibility  are  relative;  there  is  probably 
neither  absolute  immunity  nor  complete  susceptibility  to  any  infec- 
tion. There  is  furthermore,  no  hard  and  sharp  line  of  demarcation 
between  the  various  types  of  immunity;  nevertheless,  it  is  convenient 
to  assemble  the  prominent  manifestations  of  immunity  into  several 
types  or  classes. 

A.  Natural  or  Inherited  Immunity. — The  inherited  power  of  resist- 
ing specific  infection  manifested  by  a  large  proportion  of  the  individuals 
comprising  a  family,  genus  or  species  is  termed  inherited  or  natural 
immunity.     It  may  be: 

1.  Racial. — Observed  in  specific  families,  genera  or  species  of  the 
animal  kingdom,  or 

2.  Individual. — Observed  in  individuals  of  the  same  species.    Indi- 
vidual natural  immunity  may  also  be  sexual — observed  in  males  or 
females  of  the  same  species. 

B.  Acquired  or  Induced  Immunity. — The  resistance  or  non-sus- 
ceptibility to   infection  following  naturally  acquired   or  artificially 
induced  specific  diseases,  or  resistance  passively  brought  about  by  the 
introduction  of  specific  protective  substances  is  termed  acquired  or 
induced  immunity. 

1.  Active  Immunity. — (a)  Natural. — Following  naturally  acquired 
disease,  as  for  example,  immunity  following  recovery  from  smallpox 
or  typhoid  fever. 

(b)  Artificial. — Brought  about  by  the  introduction  of  attenuated 
or  killed  viruses,  vaccines  or  toxic  products  of  bacteria  into  a  sus- 
ceptible host.  The  toxic  products  of  bacteria  may  be  either  those 
8 


114  IMMUNITY  AND  INFECTION 

excreted  during  life,  or  products  arising  from  their  disintegration. 
Immunity  to  smallpox  following  vaccination  and  immunity  to  typhoid 
fever  following  the  injection  of  killed  cultures  of  typhoid  bacilli  are 
familiar  examples  of  this  type  of  immunity. 

There  is  usually  a  period  of  increased  susceptibility  to  infection 
immediately  after  the  introduction  of  the  virus  or  its  products,  in 
artificially  acquired  immunity.  This  period  of  susceptibility  is  fol- 
lowed by  an  increase  in  resistance  to  the  virus.  If  the  process  of 
immunization  is  repeated  several  times,  the  initial  level  of  resistance 
to  infection  may  be  raised  very  materially.  Thus,  prophylactic  vac- 
cination with  killed  typhoid  bacilli  (anti-typhoid  vaccination)  increases 
the  resistance  of  the  recipient  of  the  vaccine  to  typhoid  infection  to 
such  a  degree  that  his  chances  of  acquiring  the  disease  are  greatly 
lessened.  It  is  also  probable  that  in  the  event  of  infection  of  the 
protected  individual  with  the  typhoid  bacillus,  both  the  duration  and 
severity  of  the  attack  will  be  diminished. 

2.  Passive  Immunity. — (a)  Antibody  Immunity. — Introduction  into 
the  host  of  specific  products  of  immunity  (antibodies)  as  diphtheria 
antitoxin. 

(6)  Chemotherapy. — The  use  of  chemicals  for  preventing  or  modifying 
infection. 

Passive  immunity  is  induced  by  the  injection  of  antibodies  into 
the  host,  which  have  been  developed  in  another  animal.  The  recipient 
of  these  antibodies  is  protected  only  so  long  as  they  remain  in  the 
body.  The  immunity,  however,  is  effective  almost  immediately  after 
injection;  there  is  no  latent  period. 

3.  Mixed,  Active  and  Passive  Immunity. — Mixed  artificially  acquired 
immunity  is  induced  by  the  simultaneous  injection  of  specific  anti- 
bodies and  the  weakened  or  attenuated  virus;  resistance  to  infection 
is  usually  increased  at  once  (passive  immunity),  while  at  the  same 
time  the  host  begins  to  react  to  the  virus  and  to  produce  antibodies 
thereto  (artificially  acquired  immunity). 

The  factors  which  predispose  the  host  to  or  protect  him  from  inva- 
sion by  microorganisms  are  usually  varied  and  complex.  Relatively 
simple  explanations  of  the  mechanism  involved  suffice  to  account  for 
the  phenomenon  in  specific  instances,  however.  For  example,  frogs 
and  hens  are  not  naturally  susceptible  to  infection  with  the  anthrax 
bacillus,  whose  optimum  temperature  of  growth  is  37°  C.,  yet  infection 
could  take  place  if  the  body  temperature  of  either  animal  were  brought 
to  this  level,  as  Pasteur  showed  nearly  two  decades  ago.  A  change 


INFECTION  115 

in  environment  may  predispose  to  infection;  the  carnivora  in  their 
native  state  are  quite  resistant  to  infection  with  the  tubercle  bacillus, 
whereas  in  captivity  they  may  succumb  readily.  Similarly,  man 
placed  in  bad  hygienic  surroundings  appears  to  be  distinctly  more 
vulnerable  to  many  infectious  diseases  than  he  is  when  his  environ- 
ment is  more  sanitary.  Unhygienic  conditions,  however,  are  rela- 
tively complex  in  their  reactions  on  man,  for  the  attendant  evils  of 
overcrowding,  underfeeding  and  increased  exposure  to  infection 
undoubtedly  play  a  part. 

Heredity  also  appears  to  be  an  important  factor  in  determining  the 
average  severity  of  infection  in  certain  types  of  endemic  disease. 
Measles  is  a  common  and  usually  fairly  mild  disease  of  childhood 
among  civilized  people.  Among  aboriginal  populations,  as  those  of 
the  South  Sea  Islands  where  the  inhabitants  had  not  been  exposed 
to  measles  previous  to  the  advent  of  Europeans,  the  introduction 
of  the  virus  has  resulted  in  a  veritable  plague  during  which  large 
numbers  of  the  people  died.  This  phenomenon  of  hereditary  acquired 
tolerance  for  specific  endemic  disease  may  conceivably  be  even  more 
specific;  for  example,  strains  of  a  given  organism  might  produce 
mild  disease  in  areas  where  it  has  been  endemic  for  generations  and 
yet  be  rapidly  fatal  for  alien  populations  who  may  have  in  turn  become 
partly  tolerant  for  other  strains  of  the  same  organism.  If  such  prove 
to  be  the  case,  unrestricted  emigration  may  lead  to  temporary  dis- 
turbances in  the  balance  between  specific  microorganisms  on  the  one 
hand  and  hosts  on  the  other — a  feature  which  Theobald  Smith  called 
attention  to  many  years  ago.1 

Racial  differences  in  susceptibility  are  occasionally  met  with  even 
in  the  same  species.  Negroes  and  Indians  are  more  susceptible  to 
infection  with  tubercle  bacillus  than  the  Caucasian  race.  The  Jews 
appear  to  be  somewhat  more  resistant  to  infection  with  the  tubercle 
bacillus  than  the  other  branches  of  the  Caucasian  race. 

H.  INFECTION. 

Pathogenic  bacteria  which  reach  the  host  do  not  necessarily  incite 
disease;  they  may  be,  and  undoubtedly  are,  frequently  overcome  by 
the  body  without  inducing  symptoms.  This  initial  resistance  to  infec- 
tion involves  an  initial  struggle  between  host  and  microorganism  which 
brings  into  play  non-specific  lines  of  defense  of  the  macroorganism 

1  Theobald  Smith,  Tr.  Assn.  Am.  Phys.,  1893. 


116  IMMUNITY  AND  INFECTION 

consisting  collectively  of  the  skin,  mucous  membranes  of  the  respira- 
tory and  gastro-intestinal  tracts  and  other  intact  barriers  discussed 
in  the  preceding  chapter.  If  this  initial  line  of  defense  holds,  the  host 
overcomes  the  prospective  invader  and  the  latter  frequently  perishes. 
Repeated  microbic  assaults  may  be  successful  if  the  first  fails.  On 
the  other  hand,  if  the  microbe  prevails  and  penetrates  the  initial  line 
of  defense,  invasion  of  the  tissues  of  the  host  occurs  and  the  micro- 
organism encounters  a  second  line  of  defense  which  is  made  up  of 
two  rather  distinct  factors — cellular  and  humoral.  The  cellular 
defense  of  the  host  resides  in  the  leukocytes  which  circulate  in  the 
the  body  fluids  and  in  certain  fixed  tissue  cells  in  the  lungs,  lymph- 
spaces  and  glands,  the  Kupfer  cells  of  the  liver,  as  well  as  large  cells 
which  appear  in  serous  cavities.  These  cells  engulf  and  destroy 
certain  types  of  invading  microorganisms.  The  humoral  defense 
resides  in  the  natural,  non-specific  power  of  the  blood  and  lymph  to 
destroy  limited  numbers  of  microorganisms  or  to  so  interfere  with 
their  nutrition  or  other  functions  as  to  prevent  their  development 
within  the  body.  The  humoral  defense  is  frequently  effective  against 
bacteria  which  do  not  succumb  to  the  cellular  defense  of  the  body, 
and  vice  versa. 

It  is  recognized  that  certain  environmental  factors  predispose  to 
infection.  Thus,  extreme  climates,  excessive  humidity,  or  exposure 
to  unhygienic  conditions,  bad  air,  poor  or  insufficient  food,  lack  of 
exercise  or  fatigue  may  react  upon  the  individual  in  ways  not  defi- 
nitely understood  and  reduce  his  resistance  to  microbic  invasion  on 
the  one  hand,  and  his  ability  to  rally  his  specific,  anti-microbic 
mechanism  on  the  other  hand.  Intracurrent  disease  frequently 
weakens  the  initial  lines  of  defense,  permitting  bacteria  of  the  "  oppor- 
tunist" type  to  become  invasive.  Thus,  furunculosis  frequently  is 
a  complication  of  diabetes,  pneumonia  not  uncommonly  terminates 
a  case  of  tuberculosis.  Renal  and  cardiac  disease  may  weaken  the 
normal  barriers  of  the  body,  permitting  a  variety  of  infections  with 
endogenous  bacteria. 

It  is  a  well-attested  fact  that  certain  occupations  or  professions 
cause  or  promote  pathological  conditions  which  predispose  to  infec- 
tion. Prominent  among  these  is  participation  in  arts  or  industries 
which  involve  exposure  to  poisonous  or  irritating  dust  or  fumes.  The 
incidence  of  tuberculosis  among  those  frequently  exposed  to  organic 
or  inorganic  dust  is  a  striking  example  of  the  relation  of  occupation 
to  infection. 


THEORIES  OF  IMMUNITY  117 

When  an  invading  microorganism  has  reached  a  suitable  atrium  of 
the  body,  overcome  the  initial  defense  of  the  host  at  that  point,  and 
has  successfully  resisted  the  normal  humoral  or  cellular  opposition 
of  the  host,  a  new  phase  of  the  struggle  becomes  prominent,  during 
which  the  host  gradually  develops  a  specific  attack  upon  the  invader, 
bringing  into  action  latent  forces  which  constitute  the  third  and  last 
defense  of  the  body.  The  invader  also  may  change  its  weapons  to 
some  degree  to  meet  the  antimicrobic  activity  of  the  host  and  the 
result  of  the  struggle  may  be  complete  recovery  from  infection,  chronic 
disease,  the  bacillus  carrier  state,  or  death  of  the  host. 

The  initial  and  secondary  defensive  powers  of  the  host,  therefore, 
are  both  cellular  and  humoral  in  character.  The  intact  skin  and 
mucous  membranes  of  the  gastro-intestinal,  respiratory  and  genito- 
urinary tracts  are  important  initial  non-specific  lines  of  defense.  The 
phagocytic  activity  of  leukocytes  and  certain  fixed  tissue  cells,  and 
the  natural,  normal  bactericidal  substances  of  the  blood  and  lymph, 
which  bathe  the  initial  line  of  defense,  are  important  adjuvants  in 
maintaining  the  integrity  of  these  initial  barriers  to  infection.  In 
certain  infections  the  humoral  factors  are  the  more  important,  while 
in  others  the  cellular  mechanism  is  conspicuous. 

The  defensive  mechanism  against  the  same  bacterium  may  be 
different  in  one  or  another  animal.  For  example,  dogs  and  rats  are 
relatively  immune  to  infection  with  the  anthrax  bacillus.  The  immu- 
nity observed  in  the  dog  appears  to  be  due  to  phagocytic  activity  of 
leukocytes  which  engulf  and  destroy  anthrax  bacilli  which  may  have 
gained  entrance  to  the  body.1  The  rat,  on  the  contrary,  enjoys  immu- 
nity not  because  its  leukocytes  engulf  and  destroy  anthrax  bacilli; 
the  blood  of  the  rat  possesses  soluble,  non-specific  bactericidal  sub- 
stances which  destroy  anthrax  bacilli.  Frequently  both  the  cellular 
and  humoral  elements  are  engaged  either  simultaneously  or  succes- 
sively as  the  struggle  between  host  and  invading  organism  proceeds. 

m.     THEORIES   OF  IMMUNITY. 

Two  distinct  explanations  have  been  advanced  to  account  for  the 
mechanism  of  immunity  as  it  is  observed  during  the  course  of  disease : 
the  cellular  or  phagocytic  theory  championed  by  Metchnikoff  and 
his  followers,  and  the  humoral  theory  developed  by  Ehrlich. 

Both  of  these  theories,  the  cellular  and  the  humoral,  have  in  com- 

1  Hektoen,  Jour.  Am.  Med.  Assn.,  1906,  xlvi,  1407. 


118  IMMUNITY  AND  INFECTION 

mon,  tacitly  at  least,  two  important  features:,  the  specificity  of  the 
protective  substances  (antibodies)  formed  as  the  result  of  infection, 
and  the  principle  that  no  new  mechanism  is  evolved  de  novo  to  meet 
the  conditions  existing  during  an  infection;  rather,  there  is  an  increase 
in  activity  along  definite  lines  in  the  preexistent,  latent  or  reserve 
mechanism  of  defense. 

Neither  theory  affords  a  satisfactory  explanation  of  all  the  features 
of  immunity  following  infection  and  it  is  very  probable  that  cellular 


911- 


FIG.  5. — Side-chains,  first  order  (antitoxins  and  antif erments) .  1,  side-chain  attached 
to  cell;  c,.haptophore  group;  2,  side-chain  to  which  is  attached  a  toxin  molecule;  3, 
a  cast  off  side-chain  of  the  first  order:  antitoxin  or  antif erment ;  4,  a  toxin  or  enzyme 
molecule;  a,  toxophore  group;  6,  haptophore  group;  5,  a  toxoid:  the  toxophore  group 
is  destroyed,  leaving  the  haptophore  group  (6)  intact;  6,  a  toxin  molecule  attached  to 
a  cast-off  side-chain  (antitoxin),  illustrating  the  neutralization  of  toxin  by  antitoxin  in 
the  blood  stream. 

activity  and  the  production  of  specific  antibodies  is  more  important 
in  certain  types  of  infection,  while  phagocytic  activities  are  more 
intimately  concerned  in  other  types. 

A.  The  Humoral,  Side-chain  or  Ehrlich  Theory  of  Immunity.— 
According  to  Ehrlich's  conception,  every  cell  of  the  body  has  two 
functions:  a  physiological  function,  which  constitutes  a  special  type 
yt/bf  activity  of  the  cell — secretory  for  a  glandular  cell,  contractile  for 
Vv  a  muscle  cell,  or  conductive  for  a  nerve  cell — and  a  nutritional  func- 
tion, which  is  concerned  with  the  removal  of  the  necessary  food  sub- 
stances from  the  general  supply  circulating  in  the  blood  or  lymph 


THEORIES  OF  IMMUNITY 


119 


channels,  and  the  appropriation  and  eventual  utilization  of  these 
specific  food  materials  by  the  cell.  These  nutritional  substances 
undoubtedly  serve  two  purposes:  Structural,  to  replace  cellular 
waste,  and  Fuel,  to  supply  cellular  energy. 

The  nutritional  requirements  of  the  individual  cell  are  varied  as 
their  activities  are  varied,  and  Ehrlich  conceives  that  each  cell 
possesses  a  number  of  chemical  affinities  or  receptors,  for  convenience 
of  discussion  designated  as  "side-chains"  or  "haptines,"  which  are 


FIG.  6. — Side-chains,  second  order  (agglutinins  and  precipitins) .  1,  side-chain  attached 
to  cell;  c,  haptophore  group;  b,  zymophore  group  (agglutinophore  or  precipitinophore 
group);  2,  side-chain  to  which  is  attached  a  bacterial  cell;  a,  haptophore  group  of 
bacterial  cell;  3,  a  cast-off  side-chain  of  the  second  order,  agglutinin  or  precipitin;  4, 
a  side-chain  attached  to  a  bacterial  cell  (agglutination);  5,  a  bacterial  cell;  a,  hapto- 
phore group;  6,  an  agglutinoid;  the  zymophore  group  is  destroyed,  leaving  the  hapto- 
phore group  intact. 

\ 

the  means  of  attaching  to  the  cell  by  chemical  union,  the  essential 
nutritive  substances  preparatory  to  their  assimilation.  When  the 
particular  food  attached  to  the  cell  by  chemical  affinity — anchored 
by  the  side-chain,  to  use  Ehrlich's  terminology — has  been  assimilated, 
more  of  the  same  kind  of  food  is  removed  from  the  blood  stream  and 
attached  to  the  cell,  in  accordance  with  its  normal  physiological 
requirement.  The  cell,  acting  through  its  side-chain,  does  not  exhibit 


120  IMMUNITY  AND  INFECTION 

discrimination  between  nutritive  substances  and  irritating  or  harmful 
substances  which  may  accidentally  possess  the  same  combining 
affinity  for  the  cell.  Consequently,  when  poisonous  substances  pos- 
sessing chemical  affinities  similar  to  those  of  the  normal  food  sub- 
stances circulate  in  the  blood  stream,  they  may  become  attached  to 
the  cell  in  place  of  the  normal  physiological  nutrients.  The  anchoring 
of  these  poisonous  substances,  unlike  the  attachment  of  normal 
nutrient  substances,  is  followed  by  damage  to  the  cell,  or,  in  extreme 
cases,  by  the  death  of  the  cell.1  If  the  cell  is  not  actually  killed  by  the 
presence  of  the  toxic  substance  acting  upon  it  through  the  side-chains, 
it  is  irritated,  as  it  were,  and  the  toxic  substance  imposes  a  twofold 
burden  upon  the  cell — loss  of  the  side-chains  to  which  it  is  attached 
and  which  are  essential  to  maintain  the  nutrition  of  the  cell,  and 
greater  or  lesser  damage  to  the  function  of  the  cell,  due  to  toxic  inhibi- 
tion of  its  normal  activities.  A  cell  cannot  disembarrass  itself  of  the 
poison,  nor  can  it  assimilate  it.  It  can,  however,  throw  off  the  side- 
chain  with  the  poison  still  firmly  united  to  it  chemically;  the  extruded 
poison  cannot  enter  into  chemical  combination  with  other  cells  pos- 
sessing the  same  chemical  affinity,  for  it  is  already  attached  to  a  side- 
chain.  Its  combining  power  is  saturated. 

Side-chains  are  a  necessity  to  the  cell,  however;  without  them 
the  cell  would  starve.  Consequently  the  cell  regenerates  new  side- 
chains  of  precisely  the  same  kind  to  replace  those  thrown  off  after 
being  bound  to  non-assimilable  substances.  If  enough  of  the  soluble 
poison  or  toxin  circulates  in  the  blood  stream,  this  process  of  union 
of  toxin  to  the  cell  by  its  side-chains  and  its  expulsion  from  the  cell 
with  the  side-chains  attached  to  it  is  so  frequently  repeated  that  the 
cell  regenerates  side-chains  in  excess  of  the  normal  requirements,  in 
accordance  with  the  Weigert  theory  of  overproduction.  This  casting 
off  of  supernumerary  side-chains  is  important.  Were  they  not  cast 
off  the  cell  would  be  vulnerable  to  toxin  in  direct  proportion  to  the 
extra  number  of  side-chains,  which  would  furnish  extra  bonds  for  its 
attachment.  As  the  cast-off  side-chains  circulate  in  the  blood  stream, 
however,  they  are  an  element  of  protection  to  the  cell,  for  they  retain 
their  original  combining  power  for  the  toxin  and  unite  with  it  and 
neutralize  it  as  it  circulates  in  the  blood  stream;  that  is,  before  it 
can  reach  the  cell  itself.  It  will  be  seen,  therefore,  that  the  same 

1  If  the  toxic  material  circulates  in  the  blood  stream  but  does  not  become  attached 
to  the  body  cells,  it  is  harmless  to  the  host,  according  to  this  theory,  and  the  host  is 
naturally  immune. 


THEORIES  OF  IMMUNITY 


121 


mechanism  of  the  living  body  which  is  susceptible  of  being  poisoned 
becomes  the  protective  agent  if  it  circulates  in  the  blood  stream.  It 
is  obvious  that  the  cast-off  side-chains  constitute  antitoxin.  The 
body  as  a  whole  is  qualitatively  the  same  after  as  before  these  side- 
chains  are  formed  in  excess  of  the  normal  cellular  needs;  the  difference 
is  a  quantitative  one.  An  animal  is  naturally  immune,  according  to 


FIG.  7. — Side-chains,  third  order  (bacteriolysins,  hemolysins  and  cytolysins).  1, 
side-chain  attached  to  cell;  c,  haptophore  group;  b,  complementophile  group;  2,  side- 
chain  to  which  is  attached  a  bacterial  cell  (6)  and  complement  (5) ;  3,  a  cast-off  side- chain 
of  the  third  order;  amboceptor;  4,  a  cast-off  side-chain  to  which  are  attached  a  bacterial 
cell  (6)  and  complement  (5)  illustrating  lysis;  5,  complement. 

this  theory,  if  the  cells  of  the  body  do  not  unite  with  toxin,  that  is, 
if  they  do  not  contain  side-chains  which  fit  the  toxin  "  as  a  key  fits  a 
lock,"  to  use  Emil  Fischer's  analogy.  Toxin  may  circulate  in  the 
blood  stream  of  such  animals,  but  it  does  not  unite  with  the  cells. 

Side-chains  of  the  First  Order. — From  the  standpoint  of  the  side- 
chain  theory,  the  toxin  molecules  consist  of  two  groups-^a  combining 


122  IMMUNITY  AND  INFECTION 

or  haptophore  group,  and  a  poisoning  or  toxophore  group.  The  former 
is  relatively  thermostabile,  the  latter  thermolabile.  If  toxin  is  heated 
to  70°  C.  for  a  few  minutes,  or  allowed  to  stand  for  several  weeks,  it 
will  be  found  that  the  poisonous  property  of  the  toxin  has  disappeared, 
or  has  been  materially  reduced.  It  still  retains  its  original  powder  of 
uniting  with  and  neutralizing  antitoxin,  however.  The  thermolabile 
toxophore  group  has  been  destroyed  or  weakened  by  the  heating 
process,  or  on  standing.  The  thermostabile  group — the  haptophore 
group — has  not  been  impaired.  Toxin  which  has  lost  part  or  all  of 
its  original  poisoning  properties,  but  which  still  unites  with  antitoxin 
is  called  toxoid. 

The  soluble  toxins  of  the  diphtheria  and  tetanus  bacilli  are  not 
simple  substances;  they  contain  at  least  two  physiologically  separate 
poisons.  Thus,  the  toxin  of  the  diphtheria  bacillus  contains  in  addi- 
tion to  the  poison  which  produces  acute  symptoms,  a  second  poison 
which  acts  slowly  and  appears  to  be  responsible  for  postdiphtheritic 
paralyses  and  emaciation.  This  second  poison  has  less  affinity  for 
antitoxin  than  the  acute  poison,  and  it  is  called  a  toxone.  Similarly, 
the  tetanus  toxin  appears  to  consist  of  at  least  two  distinct  poisons 
— tetanospasmin,  which  has  an  especial  affinity  for  nerve  cells  and 
which  elicits  the  acute  symptoms  of  tetanus,  and  tetanolysin,  which 
causes  hemolysis  of  erythrocytes.  The  injection  of  soluble  or  exo- 
toxins  produced  by  bacteria  leads  to  the  formation  of  soluble  specific 
antibodies  which  are  called  antitoxins.  Antitoxins  are  supernumerary 
side-chains  which  have  been  produced  in  excess  of  the  physiological 
needs  of  the  cell,  in  response  to  the  stimulus  of  a  specific  toxin,  and 
cast  off  into  the  blood  stream. 

It  has  been  shown  that  repeated  injections  of  solutions  containing 
active  enzymes — as,  for  example,  rennin — into  animals,  is  followed 
by  the  appearance  in  the  blood  stream  of  specific  antibodies  which 
will  prevent  the  activity  of  the  homologous  enzyme.  These  anti- 
bodies, or  anti-enzymes,  as  they  are  called,  exhibit  the  specificity  and 
other  characteristics  which  distinguish  antitoxins. 

Antitoxins  and  anti-enzymes  are  called  side-chains  of  the  first 
order  by  Ehrlich.  They  possess  the  property  of  combining  with  and 
neutralizing  their  respective  toxins  or  enzymes. 

Side-chains  of  the  Second  Order.— If  substances  of  greater  com- 
plexity than  those  just  described  are  needed  for  the  nutrition  of  the 
cell,  some  preliminary  treatment,  probably  in  the  nature  of  digestion, 
may  be  required  to  prepare  these  substances  for  assimilation  after 


THEORIES  OF  IMMUNITY  123 

they  are  bound  to  the  cell.  A  side-chain  of  the  first  order,  which 
possesses  simply  a  combining  group,  does  not  provide  the  requisite 
power  of  digestion,  according  to  Ehrlich,  and  to  effect  this  digestion 
side-chains  of  somewhat  more  complex  structure  are  required.  Side- 
chains  of  this  more  complex  type,  side-chains  of  the  second  order, 
possess  not  only  a  combining  group  for  the  foodstuff,  but  a  digestive 
group  as  well.  This  digestive  or  zymophore  group,  as  it  is  called,  acts 
upon  foodstuffs  after  they  are  anchored  to  the  cell  by  the  combining 
or  haptophore  group.  The  complete  side-chain  of  the  second  order, 
therefore,  is  composed  essentially  of  a  combining  or  haptophore  group, 
and  a  zymophore  group  as  well.  The  haptophore  group  of  the  second 
order  side-chain  is  relatively  stabile,  but  the  zymophore  group  is 
labile  and  readily  becomes  inactive  without,  however,  impairing  the 
original  combining  ability  of  the  side-chain.  Side-chains  of  the  second 
order  are  as  vulnerable  to  pathological  substances  possessing  the 
requisite  chemical  affinity  as  side-chains  of  the  first  order,  and  repeated 
irritation  of  a  cell  by  such  pathological  substances  leads  eventually 
to  an  overproduction  of  side-chains  of  the  second  order  and  an 
elimination  of  the  supernumerary  side-chains  in  excess  of  the  physio- 
logical need  of  the  cell  into  the  blood  stream.  Side-chains  of  the 
second  order  which  are  thus  cast  off  from  the  cell  in  response  to  the 
stimulation  of  bacterial  or  other  alien  protein  are  of  importance 
immunologically.  If  the  serum  of  an  animal  containing  such  side- 
chains  is  brought  into  contact  with  a  suspension  of  the  homologous 
bacterium,  the  organisms  are  sooner  or  later  clumped  together  or 
agglutinated.  If,  on  the  contrary,  the  serum  is  brought  into  contact 
with  a  clear  solution  of  the  homologous  protein,  a  precipitate  forms. 
These  reactions  are  highly  specific  and  those  side-chains  which  cause 
agglutination  of  the  specific  bacterium  pr  precipitation  with  the 
homologous  protein  solution  are  called  respectively,  agglutinins  and 
precipitins. 

The  relative  instability  of  a  zymophore  group  of  a  side-chain  of  the 
second  order  may  be  inferred  from  the  following  experiment: 

A  serum  obtained  by  injecting  a  horse  with  repeated  graduated 
doses  of  typhoid  bacilli  will  clump  or  agglutinate  the  specific  organism 
in  high  dilution.  If  the  serum  is  heated  to  60°  or  70°  C.  for  a  few 
minutes,  or  if  it  has  been  kept  for  a  long  time,  it  will  no  longer  clump 
the  bacilli,  or,  at  least,  it  will  clump  them  imperfectly.  If  such  a  serum 
is  allowed  to  stand  in  contact  with  typhoid  bacilli  for  an  hour  or  two 
then  removed  by  centrifugalization,  it  will  be  found  that  the  bacilli 


124  IMMUNITY  AND  INFECTION 

will  no  longer  agglutinate  with  a  fresh,  highly  potent  agglutinating 
serum.  The  bacteria  are  saturated  with  the  combining  group  of  the 
serum  whose  agglutinophore  group  had  been  inactivated  by  heating. 
This  experiment  shows  that  the  combining  group  is  relatively  stabile, 
and  that  it  is  active  even  though  the  zymophore  group  is  inactive. 
A  side-chain  of  the  second  order  which  has  lost  its  ability  to  cause 
agglutination  with  a  specific  organism,  but  which  still  retains  its 
combining  power,  is  called  an  agglutinoid.  It  bears  a  striking  resem- 
blance to  a  toxoid  in  that  the  active  or  ergophore  group  is  destroyed, 
but  the  combining  group  remains  intact. 

Sera  containing  specific  precipitins  readily  lose  their  ability  to  form 
precipitates  with  the  homologous  protein.  The  precipitins  have 
changed  to  precipitinoids,  due  to  a  functional  loss  of  their  precipitino- 
phore  group. 

The  part  played  by  side  chains  of  the  second  order,  agglutinins  and 
precipitins,  in  immunity  is  not  well  understood.  Their  relation  to 
immunity  is  less  clear  than  the  relation  of  antitoxin  to  immunity. 

Side-chains  of  the  Third  Order. — Nutritive  substances  of  large  mole- 
cular aggregation  may  require  considerable  modification  to  fit  them 
for  cellular  assimilation.  Such  substances  are  removed  from  the 
blood  stream  and  bound  to  the  cell  by  side-chains  of  the  third  order. 
They  are  then  acted  upon  by  an  enzyme  (complement)  which  is 
also  present  in  the  blood  stream.  It  will  be  seen  that  both  the  nutri- 
tive element  and  a  digestive  enzyme  circulate  in  the  blood,  but  that 
no  reaction  occurs  between  them  until  they  are  both  united  by  a  side- 
chain  of  the  third  order,  which  must  therefore  consist  essentially  of 
two  combining  groups.  One  of  these,  the  cytophilic  group  or  hapto- 
phore,  unites  specifically  with  the  nutritive  element.  The  other  com- 
bining or  haptophore  group,  the  complementophilic  group,  unites 
with  the  enzyme  or  complement  which  is  present  in  the  blood  stream. 

Side-chains  of  the  third  order  are  called  amboceptors  because  they 
possess  two  combining  groups.  An  excessive  irritation  of  a  cell  by  a 
substance  capable  of  uniting  with  the  cytophilic  group  of  a  side-chain 
of  the  third  order  will  lead  to  overproduction  and  elimination  of  these 
side-chains  precisely  as  toxins  lead  to  an  overproduction  of  side-chains 
of  the  first  order  (antitoxin  formation).  The  side-chains  of  the  third 
order,  furthermore,  exhibit  specificity  for  the  substance  which  led  to 
their  overproduction,  just  as  antitoxins  exhibit  specificity  for  their 
homologous  toxins. 

It  has  been  shown  that  the  zymophoric  group  of  a  side-chain  of  the 


THEORIES  OF  IMMUNITY  125 

second  order  is  permanently  a  part  of  the  structure.  The  comple- 
ment, which  is  analogous  to  the  zymophore  group  of  the  second  order, 
is  not  attached  to  a  side-chain  of  the  third  order  until  the  cytophilic 
group  of  the  latter  has  combined  with  its  antigen.  The  zymophore 
group  of  the  second  order  side-chain  is  readily  destroyed  and  it 
cannot  be  replaced.  The  zymophoric  group  of  the  third  order  side- 
chain  is  not  an  integral  part  of  the  structure,  and  it  can  be  introduced 
under  appropriate  conditions. 

Third  order  side-chains  or  amboceptors  are  cytolysins.  Those 
specific  for  bacteria  are  called  bacteriolysins;  those  specific  for  blood 
are  called  hemolysins;  and  those  specific  for  the  cells  of  various  tissues 
or  organs  are  called  cytolysins. 

The  activity  of  the  lysins,  according  to  the  Ehrlich  theory,  depends 
on  the  union  of  non-specific  complement  and  a  specific  antigen  by  the 
specific  amboceptor.  A  union  of  antigen  and  amboceptor  may  take 
place  in  the  absence  of  complement,  but  a  union  of  antigen  and  com- 
plement cannot  take  place  in  the  absence  of  amboceptor.  The 
amboceptor,  like  other  haptophore  groups,  is  relatively  thermostabile. 
The  non-specific  complement  (found  in  fresh  blood  serum  from  any 
animal)  is  thermolabile  and  readily  destroyed. 

Thus  far  it  has  been  assumed  that  the  cells  of  the  body  defend 
themselves  against  toxins,  alien  protein  or  alien  cells  by  the  formation 
of  specific  antibodies  or  side-chains.  Welch1  has  made  the  important 
suggestion,  which  has  experimental  evidence  in  its  favor,  that  bac- 
teria may  also  produce  side-chains  which  are  specific  for  certain 
cells  of  the  host.  A  struggle  between  host  and  microbe,  therefore, 
would  not  be  one-sided;  a  dual  attempt  at  immunization  is  going 
on  during  a  bacterial  invasion,  in  which  the  microbe  attempts  to 
protect  itself  against  the  specific  weapons  of  the  host  as  the  host 
attempts  to  protect  itself  against  the  weapons  of  the  invading  micro- 
organism. Thus,  bacteria  grown  in  media  containing  agglutinating 
sera  gradually  lose  their  agglutinability,  but  this  acquired  loss  of 
agglutinating  power  is  not  exhibited  by  descendants  of  the  inagglu- 
tinable  strain 'grown  for  some  time  in  media  not  containing  agglutinins. 

The  side-chain  theory,  originally  formulated  to  explain  antitoxin 
immunity,  but  enlarged  in  its  scope  to  include  the  phenomena  of 
agglutination,  precipitation  and  cytolysis,  has  been  subjected  to  much 
adverse  criticism.  It  was  assumed  that  toxin  and  antitoxin,  for 
example,  united  in  simple  proportions  as  a  strong  acid  and  a  strong 

1  Huxley  Lecture,  1902. 


126  IMMUNITY  AND  INFECTION 

base  unite;  the  chemical  analogy  of  toxin-antitoxin  union  to  form 
an  inert  mixture  comparable  to  a  salt  was  further  accentuated  by  the 
effect  of  moderate  degrees  of  heat  in  hastening  the  reaction  between 
the  two.  A  very  thorough  investigation  of  the  quantitative  neutraliza- 
tion of  toxin  by  antitoxin  revealed  the  error  of  this  supposition  and 
Ehrlich  was  led  to  assume  a  very  complex  structure  for  the  toxin  mole- 
cule, in  which  there  existed  several  fractions  possessing  individually, 
different  affinity  for  antitoxin. 

Madsen  and  Arrhenius1  studied  the  toxin-antitoxin  union  from 
the  standpoint  of  physical  chemistry  and  found  that  the  slightly  dis- 
sociated reactive  substances  united  in  conformity  with  the  law  of 
mass  action  of  Guldberg  and  Waage.  Their  conclusion  was  that 
toxin  and  antitoxin  react  like  a  weak  acid  and  weak  base,  and  that  it 
is  a  reversible  reaction,  so  that  a  mixture  of  toxin  and  antitoxin  always 
contains  free  toxin,  free  antitoxin  and  toxin-antitoxin,  the  relative 
amounts  being  calculable  according  to  the  law  of  mass  action.  The 
observations  of  Theobald  Smith2  and  of  many  other  observers  that 
neutral  mixtures  of  toxin  and  antitoxin  would  induce  active  immunity 
in  experimental  animals  are  in  harmony  with  this-view.  Biltz3  has 
advanced  an  hypothesis,  based  upon  the  assumption  that  toxin  and 
antitoxin  are  colloids,  which  in  essence  assumes  that  the  toxin-anti- 
toxin reaction  is  a  phenomenon  of  adsorption,  quite  unlike  the  reaction 
of  a  weak  acid  and  a  weak  base. 

The  humoral  theory  of  immunity  fails  to  attribute  to  phagocytic 
cells  any  prominent  part  in  immunity.  No  theory  has  been  advanced, 
up  to  the  present  time,  which  explains  all  the  phenomena  of  humoral 
immunity;  whatever  the  final  solution  may  be,  the  side-chain  theory 
as  developed  and  defended  by  Ehrlich  must,  and  always  will  be,  a 
worthy  monument  to  a  great  man. 

B.  The  Cellular  or  Phagocytic  Theory  of  Immunity. — The  cellular 
theory  of  immunity,  formulated  and  championed  by  Metchnikoff, 
had  its  inception  in  observations  of  the  nutritive  activities  of  amebse, 
which  could  be  watched  under  the  microscope.  It  was  observed 
that  these  simple,  transparent  protozoa,  when  about  to  feed,  ap- 
proached and  flowed  around  a  minute  organism,  as  a  bacterial  cell. 
Shortly  after  engulfment  the  contour  of  the  ingested  bacterium  lying 
within  the  substance  of  the  ameba  became  less  and  less  distinct  and 

1  See  Arrhenius,  Immunochemie,  Leipzig,  1907,  for  full  details. 

2  Jour.  Exp.  Med.,  1909,  xl,  241,  Active  Immunity  Produced  by  So-called  Balanced 
or  Neutral  Mixture  of  Diphtheria  Toxin  and  Antitoxin. 

3  Ztschr.  f.  physiol.  Chem.,  1904,  615. 


THEORIES  OF  IMMUNITY 


127 


finally  disappeared  entirely.  His  attention  was  soon  directed  to  a 
small,  transparent  crustacean,  daphnia,  within  whose  body  cavity 
could  be  distinguished  minute  wandering  cells  which  exhibited  ameboid 
movements.  The  physiological  significance  of  these  ameboid  cells — 
which  are  potentially  leukocytes — was  not  clear  until  it  was  found 
that  they  engulfed  and  digested  certain  yeast  spores  that  occasionally 
gained  entrance  to  the  body  cavity  of  the  crustacean.  If  the  yeast 
spores  were  not  too  numerous  the  wandering  cells  flowed  around  and 
eventually  destroyed  them;  if,  on  the  contrary,  the  number  of  yeast 
spores  was  too  great,  the  wandering  cells  could  not  remove  the  entire 


FIG.  8. — Phagocytosis  of  gonococcus. 

number  and  the  residual  spores  germinated  and  killed  the  host.  It 
was  evident  that  the  phagocytic  activity  of  the  ameboid  cells  played 
a  prominent  part  in  protecting  daphnia  from  an  infection  with  the 
yeast. 

Next  Metchnikoff  injected  anthrax  bacilli  into  the  lymphatic  sac 
of  frogs  and  found  again  that  wandering  cells — leukocytes — engulfed 
and  destroyed  the  bacteria,  thus  preventing  infection  and  death  of 
the  frog.  This  line  of  observation  was  followed  through  an  extensive 
series  of  lower  animals,  mammals,  and  finally  in  man,  where  the 
engulfment  of  the  meningococci,  gonococci,  pneumococci,  and  staphy- 
lococci  by  polymorphonuclear  leukocytes  during  the  course  of  acute 
infections  with  these  organisms  afforded  a  striking  demonstration 


128  IMMUNITY  AND  INFECTION 

of  the  phagocytic  activity  of  leukocytes  which  circulate  normally  in 
the  blood  and  lymph  streams.  These  and  many  other  observations 
and  experiments  led  to  the  formulation  of  the  phagocytic  theory  of 
immunity.  Natural  immunity,  according  to  this  theory,  is  leukocytic 
immunity — that  is,  the  natural  barriers  of  the  body,  reenforced  by 
the  activity  of  leukocytes  in  the  blood  and  lymph  streams  which 
bathe  the  intact  skin,  mucous  membranes,  etc.,  suffice  to  protect 
the  body  against  invasion  by  moderate  numbers  of  bacteria  or  other 
microorganisms.  Infection  of  the  body,  according  to  this  view,  is 
attributable  to  a  failure  of  the  leukocytic  defense,  or  to  too  large 
numbers  of  invading  organisms,  or  both  factors  combined. 

Metchnikoff  classified  phagocytic  cells  of  the  body  into  two  groups : 

1.  .Macrocytes  or  Macrophages. — Large  mononuclear  cells  and  certain 
fixed  tissue  cells,  particularly  of  the  spleen,  liver,  lungs,  and  lymph 
nodes.     Macrophages  are  active  in  the  removal  of  necrotic  tissue, 
injured  blood  cells,  and  similar  abnormal  cellular  elements  of  the 
body,  and  in  chronic  bacterial'  infections,  notably  in  tuberculosis, 
leprosy,   and   actinomycosis.     They   contain   a   digestive   enzyme — 
macrocytase — which  dissolves  or  digests  these  abnormal  cells. 

2.  Microcytes  or  Microphages. — Chiefly  polymorphonuclear  leuko- 
cytes which  occur  in  the  blood  stream.     They  engulf  bacteria  and 
similar  cells.    Microcytes  contain  a  digestive  enzyme — microcytase — 
which  dissolves  or  digests  bacteria. 

The  substance  which  Ehrlich  regards  as  complement  is  normally 
present  in  the  leukocytes  as  macro-  and  microcytase,  according  to 
Metchnikoff.  These  cytases  are  liberated  into  the  blood  stream  when 
the  leukocytes  are  destroyed  (phagoly sis) . 

The  phenomenon  of  phagocytosis  may  be  divided  into  three  separate 
and  distinct  phases:  the  method  of  approach  of  the  phagocytic  cell 
to  its  prey  (chemotaxis),  the  engulf ment,  and  finally  the  digestion  or 
destruction  of  the  latter. 

The  Method  of  Approach. — It  was  a  matter  of  observation  by  Metch- 
nikoff and  his  followers  that  phagocytosis  was  more  marked  in  mild 
bacterial  infections  and  during  recovery  than  in  severe  infections  and 
the  early  acute  stages  of  the  disease.  The  importance  of  chemotaxis 
as  the  attractive  force  of  leukocytes  to  bacteria,  however,  was  not 
clearly  realized  until  Massart  and  Bordet1  showed  by  ingenious 
experiments  that  non- virulent  bacteria  apparently  secrete  substances 

1  Ann.  Inst.  Past.,  1891,  v,  417. 


THEORIES  OF  IMMUNITY  129 

which  draw  phagocytic  cells  to  "them.1  Virulent  organisms  of  the 
same  strain  not  only  do  not  appear  to  attract  leukocytes,  but  they 
appear  to  repel  them.  Bordet  explained  the  increase  of  virulence  of 
bacteria  through  passage  in  experimental  animals  on  the  ground  that 
the  less  virulent  individuals  were  engulfed  and  killed;  the  more  viru- 
lent members  survived  and  produced  a  thoroughly  virulent  strain. 
Yaillard  and  Vincent2  and  Vaillard  and  Rouget3  showed  that  bacterial 
toxins  may  repel  or  paralyze  leukocytic  activity;  if  tetanus  spores 
are  bathed  with  tetanus  toxin  before  injection  into  the  animal  body, 
the  leukocytes  do  not  collect  at  the  point  of  injection,  the  spores  ger- 
minate and  the  animal  dies  of  tetanus.  If,  however,  the  spores  are 
washed  free  from  tetanus  toxin  and  then  injected,  leukocytes  appear 
at  the  site  of  inoculation,  engulf  the  spores,  and  either  destroy  them 
or  prevent  their  germination. 

The  mechanism  of  chemotaxis  has  been  a  subject  of  much  discus- 
sion. Evidence  is  accumulating  which  would  suggest  that  chemo- 
tactic  stimuli  of  bacterial  origin  which  reach  leukocytes  enter  the 
phagocytic  cell  in  greater  concentration  on  that  side  which  is  nearer 
the  source  of  the  chemotactic  substance,  lowering  the  surface  tension 
at  that  point.  A  flow  of  protoplasm  in  this  direction,  in  obedience 
to  the  lowered  resistance,  will  result  in  the  protrusion  of  a  pseudo- 
podium,  which  will  continue  to  advance  until  the  surface  tension  is 
equalized.4  This  generally  occurs  when  the  leukocyte  has  flowed 
around  or  engulfed  the  organism. 

Engulfment. — The  earlier  view  associated  the  protrusion  of  pseudo- 
podia  and  the  subsequent  engulfment  of  bacteria  or  other  cell  as  an 
auto  voluntary  act  of  the  leukocyte.  The  inclusion  of  inert  particles, 
as  dust  or  other  minutely  comminuted  granules,  would  appear  to 
discredit  this  hypothesis.  The  engulfment  of  living  or  inert  bacteria 
or  other  minute  bodies  is,  as  Wells  aptly  expresses  it,5  "  but  an  exten- 
sion of  the  phenomena  of  chemotaxis.  When  the  substance  toward 
which  the  leukocyte  is  drawn  is  small  enough,  the  leukocyte  simply 
continues  its  motion  until  it  has  flowed  entirely  about  the  particle." 

Digestion. — The  ultimate  solution  of  engulfed  substances  other 
than  purely  inert  particles  is  by  intracellular  enzymes  contained  within 

1  Inert  particles,  as  coal  dust,  are  engulfed  by  phagocytic  cells;  it  is  difficult  to  explain 
this  phenomenon  on  the  basis  of  chemotaxis. 

2  La  semaine  medicale,  1891,  xi,  No.  5. 

3  Ann.  Inst.  Past.,  1892,  vi,  No.  6. 

4  See  Well's  Chemical  Pathology,  1914,  2d  ed.,  pp.  230-251    (Saunders  &  Co.),  for 
an  excellent  resume  of  the  literature. 

6  Well's  Chemical  Pathology,  1914,  2d  ed.,  p.  238  (Saunders  &  Co.). 
9 


130  IMMUNITY  AND  INFECTION 

the  phagocytic  cells.  These  enzymes  are  of  two  kinds:  macrocytase, 
present  in  the  macrophages,  and  microcytase,  found  in  the  micro- 
phages.1  Van  de  Velde,2  Buchner,3  Hahn,4  and  Bordet5  have  demon- 
strated such  endo-enzymes.  The  solution  of  bacteria  engulfed  in  leuko- 
cytes can  be  shown  by  appropriate  staining  methods;  the  organisms 
gradually  lose  their  ability  to  take  up  stain  and  eventually  disappear. 

At  this  stage  of  the  development  of  the  phagocytic  theory  of  immu- 
nity, the  important  part  played  by  the  blood  serum  in  preparing  bac- 
teria for  phagocytosis  was  prominently  set  forth  in  the  investigations 
of  Wright  and  Douglas,6  although  foreshadowed  by  the  excellent  and 
comprehensive  observations  of  Denys  and  LeClef7  and  Neufeld  and 
Rimpau.8  Wright  and  Douglas  showed  that  leukocytes,  freed  care- 
fully from  adherent  serum  by  washing  with  salt  solution,  would  not 
engulf  bacteria,  or,  at  least,  but  slowly.  The  addition  of  serum  from 
a  normal  or  immunized  animal  caused  active  phagocytosis  to  take 
place.  The  substances  in  the  blood  serum  which  prepare  bacteria 
for  engulfment  by  leukocytes  were  called  "opsonins"  by  Wright  and 
Douglas:  the  immune  opsonins — which  are  specifically  increased  in 
immunized  animals — are  almost  certainly  identical  with  the  substances 
called  bacterial  tropins  by  Neufeld  and  Rimpau.  That  the  opsonic 
substances  of  the  serum  act  primarily  upon  the  bacteria  rather  than 
upon  the  leukocytes  was  clearly  shown  by  the  observations  of  Hektoen 
and  Reudiger.9  Streptococci  suspended  in  plasma,  blood  serum  or 
defibrinated  blood  were  engulfed  by  leukocytes.  Leukocytes,  washed 
free  from  serum  or  plasma,  were  without  phagocytic  action  upon  the 
same  bacteria.  If  the  streptococci,  however,  were  allowed  to  stand 
in  contact  with  serum,  plasma,  or  defibrinated  blood  for  a  short  time 
at  37°  (a  much  longer  exposure  at  0°  to  4°  C.  was  necessary),  then 
washed  free  from  adherent  serum  or  plasma,  and  exposed  to  washed 
leukocytes,  active  phagocytosis  took  place. 

The  present  tendency  is  to  ascribe  to  phagocytosis  an  important 
part  both  in  the  destruction  of  many  kinds  of  invading  bacteria  and 
in  the  removal  of  alien  or  abnormal  cells  as  well.  The  importance 

1  For  a  detailed  discussion  of  leukocytic  enzymes,  see  Opie,  Jour.  Exp.  Med.,  1905, 
viii,  410. 

2  La  Cellule,  x,  2;  Cent.  f.  Bakt.,  1898,  xxxiii,  692. 

3  Miinchen.  med.  Wchnschr.,  1894,  718. 

4  Arch.  f.  Hyg.,  1895,  xxviii,  312. 
8  Ann.  Inst.  Past.,  1895,  ix,  398. 

6  See  Wright,  Studies  in  Immunization,  1909,  Constable. 

7  La  Cellule,  1895,  xi. 

8  Deutsch.  med.  Wchnschr.,  1904,  1458. 

9  Jour.  Inf.  Dis.,  January,  1905,  ii,  No.  1. 


THEORIES  OF  IMMUNITY  131 

of  substances  contained  within  the  plasma  or  blood  serum,  which 
prepare  bacteria  for  phagocytosis — to  use  Wright's  terminology — has 
modified  somewhat  the  original  conception  of  phagocytosis  as  proposed 
by  Metchnikoff. 

The  phagocytic  theory  and  the  humoral  theory  of  immunity  would 
appear  to  be  in  direct  opposition.  Metchnikoff  maintained  that  the 
fundamental  basis  of  immunity  resides  in  the  phagocytic  activity  of 
macro-  and  microphages.  He  believed  that  the  humoral  immune 
bodies  are  derived  either  from  leukocytes  or  the  organs  in  which 
they  are  formed — the  bone  marrow  and  lymphatic  system.  The 
champions  of  the  humoral  theory,  on  the  other  hand,  would  attribute 
the  healing  principle  to  soluble  substances  contained  in  the  body 
fluids.  The  leukocytes  and  other  phagocytic  cells,  according  to  the 
extremists  who  advocate  this  theory,  would  be  rega/ded  as  scavengers 
merely,  whose  function  it  is  to  remove  the  debris — dead  bacteria  or 
disabled  bacteria — after  they  are  overwhelmed  by  the  activity  of  the 
soluble  natural  and  immune  antibodies. 

A  final  decision  of  the  importance  of  cellular  and  humoral  factors 
in  immunity  cannot  be  made  at  the  present  time.  It  is  not  unlikely 
that  both  theories  will  be  modified  somewhat  as  additional  evidence 
accumulates. 


CHAPTER  VII. 
ANAPHYLAXIS,  ALLERGY  OR  HYPERSENSITIVENESS.1 

PROTEIN  fed  to  man  or  animals  is  reduced  to  simple  compounds, 
chiefly  amino-acids,  by  the  action  of  gastro-intestinal  enzymes  before 
it  is  absorbed  from  the  alimentary  canal.  These  gastro-intestinal 
enzymes  act  rapidly  under  normal  conditions,  and  without  an  appre- 
ciable latent  period.  One  noteworthy  result  of  digestion  is  a  complete 
denaturization  of  all  ingested  protein  before  it  enters  the  tissues  of 
the  host;  absorption  of  unaltered  or  partially-digested  protein  is 
prevented  or  reduced  to  a  minimum. 

The  importance  of  a  denaturization  of  protein  before  it  enters  the 
tissues  becomes  apparent  when  a  comparison  is  made  between  the 
effects  of  parenteral  injections  of  the  end-product^  of  prnfpin 
on  the  one  hand,  and  of  the  unaltered  protein  jtsplf  nn  tVi 
Repeated  parenteral  injections  of  amino-acids  in  moderate  amounts 
appear  to  be  without  serious  or  noteworthy  effects  upon  experimental 
animals.  A  single  parenteral  injection  of  an  unaltered  protein  is  also 
without  visible  effect,  as  a  rule.  A  second  parenteral  injection  of  the 
same  protein,  after  an  interval  of  ten  to  fourteen  days,  frequently  is 
followed  by  a  rather  definite  train  of  symptoms,  severe  in  character 
and  wholly  unlike  the  negative  response  to  a  corresponding  treatment 
with  amino-acids  or  normal  end-products  of  gastro-intestinal  digestion. 

Sensitization. — The  first  parenteral  injection  of  a  protein2  which 
is  foreign  to  the  body,  or  in  some  instances,  natural  for  the  body  but 
alien  for  the  blood,  is  without  visible  effect  upon  the  animal,  but  leads 
to  its  sensitization  to  the  specific  protein.  The  sensitizing  agent  is 
variously  referred  to  as  a  sensitizer,  sensibilisinogen,  or  anaphylac- 
togen,  and  may  be  effective  in  very  small  doses.  Rosenau  and  Ander- 
son3 were  able  to  sensitize  guinea-pigs  with  one-millionth  of  a  cubic 
centimeter  of  horse  serum;  Wells4  has  sensitized  the  same  animal 

1  For  an  excellent  resume  of  the  literature  of  anaphylaxis  complete  to   1912,  see 
Hektoen,  Jour.  Am.  Med.  Assn.,  1912,  Iviii,  1081.^ 

2  Proteins  deficient  in  tryptophane  or  tyrosin  are  said  not  to  sensitize. 

3  Bull.  29  and  36,  Hygienic  Laboratory,  Washington,  D.  C.,  1906,  1907. 

4  Wells'  Clinical  Pathology,  1914,  2d  ed.,  180. 


REINJECTION  OF  THE  HOMOLOGOUS  PROTEIN  133 

with  one  twenty-millionth  of  a  gram  of  crystallized  egg  albumen. 
Usually  0.001  to  0.1  c.c.  of  serum  is  an  effective  sensitizing  dose. 

A  latent  period  intervenes  between  the  initial  injection  of  the 
animal  with  sensitizing  protein  and  sensitization — on  the  average 
this  is  about  ten  to  fourteen  days.  Gay  and  Southard1  showed,  how- 
ever, that  the  time  necessary  to  effect  sensitization  depends  somewhat 
upon  the  size  of  the  sensitizing  dose,  larger  amounts  requiring  longer 
periods  than  smaller  amounts.  White  and  A  very2  have  found  that  a 
relation  exists  between  the  minimum  sensitizing  and  the  maximum 
intoxicating  dose,  larger  amounts  of  protein  being  required  on  rein- 
jection  to  elicit  a  reaction  when  the  sensitizing  dose  is  very  small,  and 
vice  versa. 

Reinjection  of  the  Homologous  Protein. — Repeated  injections  of  the 
homologous  protein  spaced  at  intervals  less  than  ten  days  do  not, 
as  a  rule,  cause  symptoms  of  acute  anaphylaxis — after  a  third  or  a 
fourth  injection,  however,  there  appears  at  the  site  of  the  first  injec- 
tion a  swelling,  usually  indurated  and  more  or  less  edematous,  which 
may  lead  to  extensive  necrosis  and  sloughing.  These  local  reactions, 
the  so-called  Arthus3  phenomenon,  are  closely  related  phylogenetically 
to  the  anaphylactic  symptoms  described  below. 

If  the  second  parenteral  injection  is  made  after  sensitization  is 
established — usually  after  ten  to  fourteen  days — symptoms  follow 
almost  immediately,  which  vary  somewhat  according  to  dosage  and 
the  site  of  inoculation.  A  very  large  dose  frequently  results  in  rapid 
death,  the  Theobald  Smith  phenomenon.4  Very  broadly  speaking, 
it  requires  200  to  2000  times  as  much  protein  to  cause  acute  anaphy- 
laxis as  to  effect  sensitization. 

Intravenous  or  intracerebral  injections  of  moderate  doses  are  fol- 
lowed very  soon  by  a  period  of  excitement  (in  dogs,  followed  by  a 
period  of  depression),5  the  animal  is  restless  and  moves  about  in  a 
bewildered  manner  and  shows  signs  of  respiratory  embarrassment. 
It  coughs  (a  normal  guinea-pig  rarely  or  never  coughs)  and  scratches 
the  corners  of  its  mouth.  This  state  is  followed  by  dyspnea,  with 
involvement  of  the  diaphragm  and  bronchial  musculature  leading  to 

1  Jour.  Med.  Res.,  1908,  xviii,.407. 

2  Jour.  Inf.  Dis.,  1913,  xiii,  103. 

3  Compt.  rend.  Soc.  Biol.,  1903,  Iv,  20;  1906,  Ix,  1143. 

4  Theobald  Smith,  Jour.  Med.  Res.,  1905,  xiii,  341;  Otto,  Leuthold-Gedenkschrift, 
1096,  i,  153. 

5  Guinea-pigs  in  general  react  most  strikingly  to  anaphylactic  stimuli;  man  is  less 
sensitive.     Rabbits,  sheep,  goats,  horses,  and  birds,  in  the  order  mentioned,  are  less 
susceptible  than  man.    Cold-blooded  animals  appear  to  be  refractory. 


134       ANAPHYLAXIS,  ALLERGY  OR  HYPERSENSITIVENESS 

bronchial  spasm  and  later  to  paralysis  of  respiration,1  lowered  blood- 
pressure,  frequently  cyanosis,  an4  jdeath.  Smaller  intravenous  injec- 
tions are  followed  by  the  saijfce  sympt$|Kis  of  excitement  and  respiratory 
involvement,  but  to  a  lessfc  degree,  j  Frequent  micturition  and  fluid, 
often  bloody  stools  togetheV^ItK^reat  prostration  and  dyspnea  are 
usually  observed.  The  animal  cannot  stand  and  may  die  after  several 
hours,  or  eventually  recover. 

Intraperitoneal  injections  elicit  similar  symptoms.  Subcutaneous 
injections  rarely  cause  acute  death;  as  a  rule  the  animal  has  a  febrile 
reaction  and  repeated  injections  may  be  followed  by  the  Arthus  pheno- 
menon. If  the  animal  survives  an  anaphylactic  reaction  it  is  fre- 
quently observed  to  be  more  refractory  or  even  temporarily  immune 
to  subsequent  injections  of  the  same  protein.  This  refractory  state 
is  called  anti-anaphylaxis  by  Besredka  and  Steinhardt.2  This  period 
of  refractoriness  is  of  variable  duration. 

The  postmortem  appearance  of  guinea-pigs  which  have  died  from 
the  effects  of  acute  anaphylaxis  is  usually  striking  and  characteristic. 
The  lungs  remain  fully  distended  when  the  thorax  is  opened,  the  cut 
surface  is  rather  dry,  and  death  appears  to  have  resulted  from 
asphyxiation  due  to  a  tonic  spasm  of  the  bronchial  musculature.3 

Severe  but  non-fatal  anaphylactic  reactions  are  accompanied  by  a 
lowering  of  the  body  temperature,  lowered  arterial  pressure,  leucopenia, 
frequently  with  a  temporary  partial  or  complete  loss  of  coagulability 
of  the  blood,4  followed  by  a  secondary  febrile  rise  of  temperature  and 
a  leukocytosis  in  which  polymorphonuclear  leukocytes  and  frequently 
eosinophiles5  are  increased.  Animals  killed  during  the  early  acute 
symptoms  show  but  little  distention  of  the  lungs — the  lesions  may 
resemble  those  of  an  acute  toxic  gastro-enteritis.  Ecchymoses  and 
ulcers  may  be  found  occasionally  in  the  stomach  and  intestines, 
together  with  parenchymatous  degeneration  of  the  liver  and  particu- 
larly the  kidneys,  which  may  lead  eventually  to  fatty  degeneration  of 
these  organs. 

The  symptoms  of  anaphylaxis  may  be  masked  or  even  prevented  by 
the  administration  of  certain  drugs  immediately  before  the  reinjec- 
tion — of  these  atropin,  chloral  hydrate  and  similar  narcotics  are  con- 
sidered particularly  efficient. 

1  Auer  and  Lewis,  Jour.  Am.  Med.  Assn.,  1909,  liii,  6;  Biedl  and  Kraus,  Wien.  klin. 
Wchnschr.,  1910,  844. 

2  Ann.  Inst.  Past.,  1907,  xxi,  117,  384. 

3  Auer  and  Lewis,  loc.  cit. 

4  Biedl  and  Kraus,  Wien.  klin.  Wchnschr.,  1909,  363;  Friedberger  and  Grober,  Zeit. 
f.  Immunitatsforsch.,  1911,  ix,  216. 

B  Moschowitz,  New  York  Med.  Jour.,  1911,  Ixxxxiii,  15. 


THE  NATURE  OF   THE  POISON,   ANAPHYLATOXIN         135 

THE  NATURE  OF  THE  POISON,  ANAPHYLATOXIN. 

The  anaphylactic  reaction,  like  other  serological  reactions,  appears 
to  depend  upon  the  elaboration  of  a  specific  antibody  in  the  sen- 
sitized animal.  The  specificity  of  the  reaction  is  very  striking  in  the 
physiological  sense — the  serum  of  one  animal  fails  to  sensitize  for  the 
serum  of  an  unrelated  animal.  Egg  protein  of  one  species  also  fails 
to  sensitize  an  animal  against  the  egg  protein  of  another  species. 
Osborne  and  Wells,1  using  vegetable  proteins  which  can  be  obtained 
in  a  state  of  relative  purity,  have  shown  that  sensitization,  in  the 
last  analysis,  depends  chiefly  upon  the  chemical  composition  of  the 
sensitizer.  Thus,  one  vegetable  protein  fails  to  sensitize  against  a 
second,  unlike  protein,  even  though  they  be  derived  from  the  same 
seed. 

The  specificity  of  the  reaction  is  striking — it  takes  place  only  in 
response  to  a  second  injection  of  the_jipmplogQus  protein,  but  the 
symptomatology  is  essentially  the  same,  irrespective  of  the  sensitizer. 
The  promptness  with  which  the  reaction  appears  after  the  reinjection 
suggests  at  once  that  the  poison,  is  radically  different  from  a  true 
bacterial  toxin,  which  invariably  requires  a  definite  latent,  period 
before  symptoms  can  be  detected,.]  In  this  respect  the  anaphylatoxin 
resembles  somewhat  an  alkaloidal  poison.  Up  to  the  present  time  no 
antitoxins  have  been  prepared.  The  action  of  the  poison  is  peripheral 
rather  than  central,  according  to  Auer  and  Lewis.2  Schultz3  and 
others  have  shown  that  it  acts  powerfully  upon  smooth  muscle  fibers; 
Biedl  and  Kraus4  and  others  have  shown  that  an  injection  of  peptone 
into  dogs  elicits  symptoms  and  pathological  changes  indistinguishable 
from  those  of  anaphylaxis.  They  were  inclined  to  regard  the  anaphy- 
latoxin as  similar  to,  or  possibly  identical  with  peptone.  Animals 
immune  to  anaphylactic  reactions  react  slightly  or  not  at  all  to  peptone 
injections. 

Passive  anaphylaxis  may  be  induced  in  a  non-sensitized  animal 
by  an  injection  of  the  serum  of  a  sensitized  animal.  Usually  a  few 
hours  elapse  before  the  recipient  of  the  specific  antibody  is  reactive, 
however.  The  experiments  of  Pearce  and  Eisenbrey,5  of  Weil,6  Dale,7 

1  Jour.  Inf.  Dis.,  1913,  xii,  341. 

2  Loc.  cit. 

3  Hygienic  Laboratory  Bulletin,  1912,  No.  80. 

4  Wien.  klin.  Wchnschr.,  1901,  No.  11. 

5  Journ.  Inf.  Dis.,  1910,  vii,  565. 

6  Jour.  Med.  Res.,  1913,  xxvii,  497;  1914,  xxx,  87,  299. 

7  Jour.  Pharm.  and  Exp.  Therap.,  1913,  iv.  167. 


136        ANAPHYLAXIS,  ALLERGY  OR  HYPERSENSITIVENESS 

Schultz1  and  others  indicate  that  the  reaction  occurs  within  the  cells 
of  the  body  rather  than  in  the  blood  stream.  The  urine  of  anaphy- 
lactic  animals  is  toxic  and  2  c.c.  is  frequently  sufficient  to  kill  guinea 
pigs  with  anaphylactic  symptoms,  according  to  Pfeiffer.2 
<(  Anaphylaxis  may  be  defined  as  a  congenital  or  acquired  condition 
of  hypersensitiveness  of  man  or  animals  tojthe  parenteral  introduction 
of  proteins,  which  is  incited  byjme  or  more  injections  of  Jbacterial, 
plant,  animal  or  huma_n  protein^  Active  acquired  hypersensitiveness 
can  be  transmitted  to  non-sensitized  individuals  by  the  injjectionpf 
the  serum  of  an  anaphylacticized  individual,  inducing  in  the  recipient 
of  the  serum  a  condition  of  passive  anaphylaxis.)  Anaphylaxis,  there- 
fore, belongs  to  the  group  of  immuhological  reactions. 

Theories. — Vaughan3  has  shown  that  all  proteins  may  be  split  into 
two  fractions  if  they  are  heated  with  alcoholic  potassium  hydroxide; 
one  portion,  insoluble  in  alcohol,  when  injected  into  animals  gives 
symptoms  indistinguishable  from  those  of  anaphylaxis,  irrespective 
of  the  protein.  The  alcohol-soluble  fraction  is  not  toxic.  The  alcohol- 
insoluble  fraction  obtained  from  various  animal,  vegetable,  and 
bacterial  proteins  always  reacts  the  same,  not  only  symptomatically, 
but  quantitatively  as  well.  His  theory  is  that  the  protein  molecule 
consists  of  two  parts:  an  archon  or  nucleus,  which  is  poisonous  and 
elicits  the  symptoms  of  anaphylaxis  when  it  is  injected  parenterally 
into  animals,  and  common  to  all  proteins;  and  additional  groups 
which  are  non-poisonous,  but  confer  upon  a  protein  by  their  number 
and  arrangement,  its  specificity.  When  a  protein  is  injected  paren- 
terally into  an  animal,  the  cells  of  the  animal  elaborate  an  enzyme 
which  will  specifically  disintegrate  it.  Among  the  products  of  disin- 
tegration is  the  poisonous  nucleus  or  archon  in  a  more  or  less  free 
state.  The  liberation  of  this  substance  causes  acute  poisoning  of  the 
host.  This  substance,  for  which  no  antibody  or  antitoxin  has  been 
prepared  so  far,  is  the  "endotoxin"  of  bacteria. 

Many  of  the  phenomena  of  anaphylaxis  are  readily  explained  in 
the  light  of  Vaughan's  work.  The  latent  period  or  pre-anaphylactic 
state  which  intervenes  between  the  injection  of  a  protein  and  the 
appearance  of  sensitization  is  the  time  required  to  mature  the  specific 
enzyme.  The  specificity  of  the  enzyme  (called  forth  by  the  stimulus 
of  alien  protein  in  the  tissues)  is  determined  by  the  arrangement  and 

1  Loc.  cit. 

2  Zeit.  f.  Immunitatsforsch.,  1911,  x,  550. 

3  Protein  Split  Products,  1913,  for  full  discussion. 


THE  NATURE  OF  THE  POISON,  ANAPHYLATOXIN         137 

number  of  groups  arrayed  around  the  poison  group  of  the  protein; 
the  similarity  or  identity  of  the  symptoms  of  anaphylaxis  irrespective 
of  the  protein  depends  upon  the  liberation  of  the  poison  nucleus 
(common  to  all  proteins)  in  a  relatively  free  state.  The  induction 
of  passive  sensitization  depends  upon  the  injection  of  this  specific 
enzyme,  which  is  present  in  the  serum  of  a  sensitized  animal,  into  a 
non-sensitized  animal.1 

Vaughan  regards  the  formation  of  a  specific  proteolytic  enzyme  in 
response  to  the  injection  of  alien  protein  into  the  tissues  as  a  protec- 
tive mechanism  to  rid  the  body  of  foreign  substance;  the  theoretical 
importance  of  this  conception  as  a  purposeful  reaction  is  clearly 
shown  in  bacterial  infections.  The  incubation  period  of  many  bac- 
terial infections  is  about  two  weeks,  during  which  clinical  symptoms 
are  not  pronounced.  This  is  interpreted  as  the  time  required  by  the 
cells  of  a  host  to  mature  a  specific  enzyme  capable  of  disintegrating 
the  alien  protein  (bacterial  cells).  The  symptomatology  of  bacterial 
disease  is  caused  largely  by  the  liberation  of  the  poisonous  nucleus 
of  the  bacterial  protein  in  special  tissues  or  organs.  Natural  immunity 
to  bacterial  disease,  according  to  this  theory,  is  due  to  the  inability 
of  the  organism  to  grow  in  the  tissues  of  the  host;  active  immunity 
is  conferred  on  the  host  by  the  presence  of  a  persistent  enzyme  which 
will  disintegrate  the  specific  organism  whenever  it  is  reintroduced 
into  the  body. 

Chemically,  the  poison  nucleus  or  endotoxin  is  stated  by  Vaughan 
to  resemble  beta-imidazoleethylamine,  described  previously.2  The 
specificity  of  the  anaphylactic  reaction  depends  upon  the  cleavage  of 
the  protein  molecule  by  a  specific  proteolytic  enzyme  with  the  libera- 
tion of  a  non-specific  poisonous  product  of  protein  degradation. 
Abderhalden3  and  his  associates  have  demonstrated  proteolytic 
enzymes  in  the  blood  stream. 

Friedberger4  has  shown  that  a  poison  may  be  obtained  by  incubating 
the  inactivated  serum  of  a  sensitized  animal  with  an  excess  of  com- 
plement and  homologous  (sensitizing)  protein,  which,  when  injected 
into  guinea-pigs,  elicits  the  symptoms  of  anaphylaxis.  It  is  not 
true  toxin,  for  no  antibody  is  produced  in  response  to  repeated,  sub- 
lethal  injections;  it  appears  to  differ  from  Vaughan's  poison  in  that 

1  The  importance  of  the  degradation  of  protein  in  the  alimentary  tract  can  be  appre- 
ciated in  the  light  of  what  has  been  stated  about  anaphylaxis. 

2  See  page  76. 

3  Zeit.  f.  physiol.  Chem.,  1912,  Ixxxii,  109;  Abwehrfermente  des  tierischen  Organismus, 
Berlin,  1913. 

4  Zeit.  f.  Immunitatsfbrsch.,  iv,  636;  vii,  94;  Ueber  Anaphylaxie,  Ibid.,  1911,  ix,  394 
(in  collaboration  with  Goldschmidt,  Schmanowsky,  Schiiltze,  and  Nathan). 


138        ANAPHYLAXIS,  ALLERGY  OR  HYPERSENSITIVENESS 

it  is  destroyed  or  inactivated  at  a  temperature  above  65°  C.  The 
poison  does  not  form  if  complement  is  not  present  in  solution  with 
the  inactivated  serum  and  antigen,  which  would  suggest  a  resemblance 
to  other  cytolytic  reactions  in  which  the  specific  amboceptor  is  acti- 
vated by  complement. 

The  essential  distinction  between  the  theory  of  Vaughan  and  that 
of  Friedberger  would  appear  to  rest  upon  the  nature  of  the  poisonous 
substance  liberated;  Vaughan  would  maintain  the  specificity  of  the 
enzyme  and  the  identity  of  the  poisonous  substances  formed  from 
various  proteins.  Friedberger's  theory,  which  was  developed  several 
years  after  Vaughan's  first  work,  would  emphasize  the  distinction 
between  an  enzyme  and  the  specific  amboceptor,  which  requires  com- 
plement for  its  activation.  Keysser  and  Wassermann1  and  more 
recently,  Jobling  and  Petersen2  have  found  that  serum  shaken  with 
kaolin,  chloroform,  and  other  agents  will  absorb  substances  from 
serum,  leaving  the  remainder  toxic  for  guinea-pigs;  the  reaction 
induced  by  the  injection  ef  small  amounts  of  altered  serum  resembles 
closely  that  of  anaphylaxis.  Jobling  and  Petersen  believe  that  the 
toxic  substance  originates  not  from  bacteria  necessarily,  but  from 
serum  itself.  Under  normal  conditions,  anti-enzymes  prevent  the 
normal  serums  from  causing  auto-autolysis;  kaolin,  bacteria,  etc., 
added  to  the  serum,  absorb  and  thus  remove  the  anti-enzymes,  thus 
permitting  the  serum  to  digest  itself.  In  other  words,  the  poisonous 
substance  may  originate  in  the  serum  rather  than  in  the  bacteria  or 
other  alien  protein.  These  facts  do  not  necessarily  detract  from 
Vaughan's  theory,  but  until  more  is  known  of  the  entire  subject,  a 
final  discussion  of  the  mechanism  of  anaphylaxis  must  be  postponed. 

ANAPHYLAXIS  IN  MAN. 

Natural  Hypersensitiveness. — It  has  long  been  known  that  the 
inhalation  of  organic  substances — as  the  pollen  of  various  plants,  or 
emanations  from  horses  or  guinea-pigs,  of  peptone  or  other  similar 
material — may  excite  acute  coryza  and  that  train  of  symptoms  popu- 
larly recognized  as  " hay-fever"  or  "pollen  fever"  in  some,  but  by  no 
means  all,  individuals.  If  the  specific  pollen  or  dust  is  rubbed  on  the 
nasal  mucosa  of  these  sensitized  individuals  a  violent  reaction  will 
take  place.  Other  individuals  develop  a  severe  urticaria  if  they  eat 
certain  proteins:  the  flesh  of  arthropods,  particularly  crabs  and 
lobsters,  vegetables,  eggs,  milk  are  known  to  excite  symptoms  in  indi- 

1  Ztschr.  f.  Hyg.,  1911,  Ixviii,  p.  535. 

2  Jour.  Exp.  Med.,  June,  1914,  xix,  p.  480. 


ANAPHYLAXIS  IN  MAN  139 

viduals  who  exhibit  an  "idiosyncrasy"  to  one  or  another  of  these 
substances.  This  idiosyncrasy  to  foreign  protein  may  be  either 
congenital  or  post-natal;  the  protein  is  supposed  to  have  passed 
unchanged  through  the  intestinal  tract  in  the  latter  case.  The  pheno- 
mena in  these  instances  are  explained  on  the  basis  of  sensitization 
with  specific  protein;  a  mild  anaphylactic  reaction  occurs  when 
the  specific  dust  reaches  the  nasal  mucous  membrane  or  the  specific 
protein  enters  the  digestive  tract. 

The  tendency  at  the  present  time  is  to  regard  certain  clinical  and 
pathological  symptoms  of  bacterial  infections — particularly  fever— 
and  the  production  of  specific  pathological  lesions  as  manifestations 
of  anaphylaxis  as  outlined  by  Vaughan.1  The  body  is  sensitized  to 
the  alien  protein,  be  it  organic  dust,  protein  of  the  food,  or  invasive 
bacteria;  the  anaphylactic  reaction  takes  place  when  the  homologous 
protein  is  brought  into  contact  with  the  sensitized  individuals  through 
the  proper  channels.  It  will  be  remembered  that  the  incubation  period 
in  many  bacterial  infections  was  explained  as  the  time  elapsing  between 
the  arrival  of  the  alien  protein  (bacterial  cells)  in  the  tissues  of  the 
host  and  the  maturing  of  a  specific  proteolytic  enzyme  that  would 
effect  their  disintegration.  The  symptomatology  of  bacterial  infec- 
tions, according  to  Vaughan,  is  largely  due  to  the  liberation  of  the 
anaphylatoxin  incidental  to  the  lysis  of  the  residual  organisms. 

Artificial  or  Acquired  Hypersensitiveness.  —  The  phenomena 
grouped  for  convenience  as  acquired  hypersensitiveness  are  met  with 
chiefly  in  connection  with  the  administration  of  the  sera  of  animals 
immunized  for  therapeutic  purposes.  Three  types  of  anaphylactic 
reaction  may  be  recognized : 

1.  Sudden  Death. — A  very  few  cases  are  on  record  in  which  the 
administration  of  antitoxin  for  therapeutic  purposes,  either  for 
immunization  or  curatively,  has  been  followed  within  a  few  minutes 
or  hours  by  death.  Already,  in  1896,  Gottstein2  had  collected  12 
which  followed  the  injection  of  diphtheria  antitoxin,  8  of  whom  were 
diphtheritic,  4  healthy  individuals.  About  1  in  every  50,000  appears 
to  be  the  proportion  of  deaths  due  to  an  injection  of  therapeutic  sera. 
The  symptoms  are  essentially  those  observed  in  sensitized  experi- 
mental animals  which  die  shortly  after  the  injection  of  the  homologous 
protein.  Behring,  Kitasato  and  other  observers  had  noticed  many 
years  ago,  when  antitoxin  was  first  prepared  on  a  large  scale,  that 
animals  immunized  with  large  amounts  of  tetanus  or  diphtheria  toxin 

1  Loc.  cit. 

2  Therap.  Monatschr.,  1896,  Heft  5. 


140       ANAPHYLAXIS,  ALLERGY  OR  HYPERSENSITIVENESS 

occasionally  succumbed  to  a  subsequent  small  dose  of  the  homologous 
toxin,  although  the  blood  serum  of  these  animals  contained  much 
specific  antitoxin. 

2.  Serum  Sickness  or  Serum  Disease. — Attention  was  first  directed 
to  serum  sickness  by  von  Pirquet  and  Schick,1  who  noticed  that  there 
occasionally  developed  in  individuals  who  had  received  an  injection 
of  antitoxic  sera,  usually  after  seven  to  fourteen  days,  fever  and  a 
rash  which  might  be  urticarial,  scarlatinal,  or,  in   the  more  severe 
cases,  morbilliform;  enlargement  of  lymph  glands,  particularly  those 
near  the  site  of  inoculation;  and  joint  pains,  more  frequently  of  the 
metacarpal  joints.    A  slight  edema,  frequently  of  the  angioneurotic 
type,  was    also   occasionally  observed.     The  fever  is  usually  slight 
and  there  are  signs  of  respiratory  embarrassment,   not  as  a  rule 
marked,   but  occasionally  severe.     These   reactions,   sudden  death 
and  serum  sickness,  are  more  common  in  asthmatics,  and  in  those 
individuals  presenting  the  pathological  syndrome   known  as  status 
lymphaticus. 

According  to  Moschowitz,2  these  individuals,  particularly  the 
asthmatics,  present  an  eosinophilia.  The  exact  cause  of  sudden  death 
following  the  administration  of  diphtheria  antitoxin  is  not  definitely 
known,  but  it  has  been  assumed  that  respiratory  involvement  is  a 
potent  factor.  The  appearance  of  serum  disease  seven  to  fourteen 
days  after  the  administration  of  antitoxin  is  supposed  to  depend  upon 
the  fact  that  some  of  the  alien  protein  (serum)  remains  in  the  body 
during  the  period  of  pre-anaphylaxis  (period  of  sensitization),  and 
that  this  residual  protein  is  broken  down  by  the  mature  specific  enzyme 
or  enzymes  with  the  liberation  of  a  poisonous  substance  which  causes 
the  anaphylactic  shock. 

3.  Arthus  Phenomenon. — During  the  course  of  immunization  against 
rabies  by  the  Pasteur  method  it  is  frequently  noticed  that  after  three 
or  four  injections  a  subsequent  injection  causes  symptoms  of  inflam- 
mation at  the  site  of  the  first  injection,  and  that  this  phenomenon  is 
repeated,  usually,  but  not  always,  with  diminishing  intensity  at  the 
site  of  earlier  injections  as  the  treatment  progresses.     This  inflam- 
matory reaction  at  the  site  of  injection  is  not  due  to  bacterial  infection 
ordinarily,  but  is  rather  an  expression  of  anaphylaxis.    It  is  comparable 
to  the  Arthus  phenomenon  produced  in  rabbits  by  successive  injec- 
tions of  serum  referred  to  above.     Also  in  re  vaccination  (vaccinia) 
a  so-called  accelerated  reaction  may  occur  the  second  time  the  indivi- 

1  Die  Serumkrankheit,  Leipzig,  1905. 

2  New  York  Med.  Jour.,  1911,  Ixxxxiii,  15. 


ANAPHYLAXIS  IN  MAN  141 

dual  is  vaccinated.  This  accelerated  reaction  again  is  a  mild  edition 
of  the  Arthus  phenomenon. 

4.  Prophylaxis. — At  first  sight  it  might  appear  that  the  administra- 
tion of  diphtheria  and  tetanus  antitoxin  for  therapeutic  purposes  would 
be  a  dangerous  procedure.  If  there  is  reason  to  suspect  that  the 
patient  would  react  to  the  injection  of  antitoxin  it  is  advisable  to 
inject  0.1  or  0.2  c.c.  subcutaneously  and  wait  half  an  hour.  If  no 
symptoms  develop,  the  full  dose  may  be  given  without  danger;  it 
is  generally  believed  that  even  if  mild  symptoms  do  follow  the  initial 
injection,  the  full  dose  may  be  given  with  safety  after  half  an  hour; 
the  first  injection  appears  to  abort  what  otherwise  might  be  a  reaction 
dangerous  to  the  patient 

The  present  method  of  concentrating  diphtheria  antitoxin  by  frac- 
tional precipitation  of  the  globulin1  appears  to  reduce  very  materially 
the  incidence  of  serum  sickness.  According  to  German  investigators, 
antitoxin  which  has  stood  for  one  or  two  months  has  lost  to  a  very 
considerable  extent  the  substance  or  substances  which  cause  the 
symptoms  of  serum  sickness. 

Practical  and  Theoretical  Considerations. — A.  Advantage  is  taken 
of  the  sensitization  of  individuals  by  bacterial  protein  during  certain 
bacterial  infections,  particularly  those  with  the  tubercle  bacillus, 
B.  mallei,  and  in  syphilis,  for  diagnostic  purposes.  It  has  been  shown 
almost  beyond  doubt  that  individuals  suffering  from  these  diseases  are 
sensitized  to  the  bacterial  protein,  and  it  is  possible  to  make  a  fairly  defi- 
nite clinical  diagnosis  by  introducing  extracts  of  the  specific  organisms 
into  the  skin  and  inducing  there  an  anaphylactic  reaction  which,  if  the 
dose  is  small,  is  local  in  character,  but  which  may  be  general  and  severe 
if  the  dose  is  increased  in  amount.  The  von  Pirquet,  Calmet,  Moro,  and 
Koch  methods  of  utilizing  tuberculin  for  diagnostic  purposes  are  directly 
dependent  upon  this  reaction  of  hypersensitiveness.  The  diagnostic 
use  of  mallein  and  luetin  depend  upon  the  same  phenomenon. 

B.  Advantage  is  also  taken  of  the  specificity  of  the  anaphylactic 
reaction  for  the  recognition  of  proteins.  Wells  and  Osborn2  and 
many  others  have  sensitized  guinea-pigs  with  proteins  and  then 
injected  into  these  sensitized  animals  proteins  which  are  to  be  iden- 
tified either  specifically  or  phylogenetically.  The  nature  and  extent 
of  the  anaphylactic  reaction  in  these  animals  furnishes  the  most  deli- 
cate test  (except  possibly  the  precipitin  test)  which  is  available  for 
such  investigations. 

1  Banzhof,  Johns  Hopkins  Hosp.  Bull.,  1911,  xxii,  241. 

2  Loc.  cit. 


CHAPTER  VIII. 

ANTIGENS  AND  THE  TECHNIC  OF  SERUM 
REACTIONS. 


NATURE  OF  ANTIGENS  AND  ANTIBODIES. 
AGGLUTININS  AND  PRECIPITINS. 
LYSINS. 

Hemolysis  and"  the  Complement  Fixa- 
tion Reaction. 


AGGRESSINS. 

OPSONINS,  TROPINS.      BACTERIAL  VAC- 
'CINES. 


NATURE  OF  ANTIGENS  AND  ANTIBODIES. 

THOSE  substances  which  cause  specific  antibody  formation  when 
they  are  introduced  into  the  tissues  or  the  body  fluids  of  the  host  are 
called  antigens.  Their  chemistry  is  as  yet  unknown,  but  available 
evidence  would  indicate  that  they  are  protein  in  nature  and  highly 
organized  chemically.  Degradation  products  of  proteins,  as  albu- 
moses  and  peptones  and  carbohydrates  and  fats,  are  not  ordinarily 
antigenic,  that  is,  they  do  not  lead  to  antibody  formation  when  they 
are  introduced  into  the  animal  body.1  The  antigenic  properties  of 
lipoids  are  still  a  subject  of  controversy:  lipoids  appear  to  play  a 
prominent  part  in  certain  types  of  immunological  reactions,  but  their 
ability  to  stimulate  specific  antibody  formation  cannot  be  regarded 
as  proven  at  the  present  time.2 

The  function  of  antibodies  as  specific  offensive  weapons  of  the 
host  against  alien  organisms  or  their  products  has  long  been  recog- 
nized in  bacteriology,  and  most  important  laboratory  diagnostic 
methods  have  been  elaborated  through  a  study  of  the  reactions 
between  specific  antigens  and  their  respective  antibodies.  Antibodies 
are  soluble  and  are  found  in  various  concentrations  in  blood  serum 
derived  from  immunized  animals.  Many  attempts  have  been  made 
to  determine  changes  in  the  chemical  composition  or  physical  proper- 
ties of  immune  sera  from  those  of  normal  serum.  Atkinson,3  Gibson, 

1  The  injection  of  carbohydrates  and  fats  may,  however,  lead  to  specific  enzyme 
formation.      See   Rohmann,    Antigene   Wirkung   der   Kohlenhydrate,    Deutsch.    med. 
Wchnschr.,  1914,  xl,  204. 

2  See  Pick,  Kolle,  and  Wassermann,   Handbuch  der  pathogenen  Mikroorganismen, 
2d  ed.,  Bd.  I,  for  discussion  of  the  chemistry  of  antigens. 

3  Jour.  Exp.  Med.,  1899,  iii,  649. 


AGGLUTININS.    AGGLUTINOIDS  AND  PROAGGLUTINOIDS       143 

and  Banzhaf1  and  others  have  found  that  the  sera  of  horses  immunized 
to  diphtheria  toxin  show  a  marked  increase  in  globulin  content,  with 
a  decrease  in  albumin  content.  Beljaeff2  could  find  no  appreciable 
change  in  the  refractive  index,  specific  gravity,  freezing  point  or 
reaction  of  the  serum  of  an  immune  animal  above  that  of  a  normal 
animal. 

The  chemical  nature  of  antibodies,  aside  from  their  apparently 
close  relation  to  globulin,  has  not  been  determined.  There  is  evidence 
that  antitoxin  molecules  may  be  larger  than  toxin  molecules,  how- 
ever. Martin  and  Cherry3  found  that  toxins  could  be  forced  through 
dense  porcelain  filters  impregnated  with  gelatin,  which  would  restrain 
antitoxin,  and  Arrhenius  and  Madsen4  determined  that  the  toxin  mole- 
cule diffused  several  times  as  rapidly  as  the  antitoxin  molecule,  from 
which  observation  they  assumed  that  the  antitoxin  molecule  was 
larger  than  the  toxin  molecule. 

AGGLUTININS.   AGGLUTINOIDS   AND   PROAGGLUTINOIDS. 

Gruber  and  Durham5  appear  to  have  been  the  first  to  clearly  demon- 
strate specific  clumping  in  broth  cultures  of  typhoid  and  cholera 
organisms  when  their  respective  sera  were  added  to  them.  Somewhat 
later  Widal,6  and  independently  Griinbaum,7  utilized  the  principle 
of  the  specific  agglomeration  of  bacteria  by  their  immune  sera  for 
the  diagnosis  of  typhoid  fever.  They  found  that  relatively  early  in' 
the  disease,  sera  of  typhoid  patients  clumped  typhoid  bacilli  from 
broth  cultures.  Pfaundler8  observed  that  typhoid  bacilli  grown  in 
broth  containing  low  concentrations  of  specific  sera  grew  out  into  long, 
tangled  filaments,  the  "  thread"  reaction.  Originally  this  phenome- 
non was  regarded  as  highly  specific,  but  it  has  largely  given  way  to  the 
macroscopic  or  microscopic  agglutination  test. 

Agglutination  in  the  bacterial  sense  may  be  defined  as  a  clumping 
or  agglomeration  of  bacteria  from  a  uniform  suspension  in  a  fluid 
medium,  brought  about  by  the  addition  of  specific  antibodies — 
agglutinins.  It  takes  place  in  two  stages  if  motile  bacteria  are  con- 
cerned. First  there  is  loss  of  motility — "immobilization" — and 

1  Jour.  Exp.  Med.,  1910,  xii,  411. 

2  Cent.  f.  Bakt.,  Orig.,  1903,  xxxiii,  293,  396. 

3  Proc.  Royal  Soc.,  1898,  Ixiii. 

4  Festskrift  Statens  Serum  Institute,  1902. 
6  Munchen.  med.  Wchnschr.,  1896,  No.  13. 

6  La  Semaine  Medicale,  1896,  No.  13. 

7  Brit.  Med.  Jour.,  1897,  May  1,  and  Munchen.  med.  Wchnschr.,  1897,  330. 

8  Cent.  f.  Bakt.,  1898,  xxiii,  9,  71,  131. 


144   ANTIGENS  AND  THE  TECHNIC  OF  SERUM  REACTIONS 

eventually  clumping.  Smith  and  Reagh1  working  with  a  non-motile 
hog  cholera  bacillus  have  demonstrated  both  flagella  and  somatic 
agglutinins,  the  former  paralyzing  the  activity  of  the  flagella,  the 
latter  agglomerating  the  organisms  themselves.  Non-motile  bacteria 
usually  agglutinate  somewhat  more  slowly  than  motile  organisms. 
Small  amounts  of  neutral  salts  are  necessary  for  the  clumping  of 
bacteria,2  although  a  union  of  the  specific  organism  and  its  agglutinin 
will  take  place  even  if  salts  are  absent.  The  specific  substance  (or 
substances)  of  the  bacterial  cell  which  reacts  with  the  specific  antibody 
of  the  serum  (agglutinin)  is  known  as  agglutinogen.  Closely  related 
bacteria,  as  typhoid  and  paratyphoid  bacilli,  may  possess  a  certain 
amount  of  agglutinogen  in  common,  but,  as  a  rule,  the  specific 
organisms  are  clumped  in  immune  sera  at  much,  greater  dilution  than 
related  organisms  are  clumped.  Also,  the  specific  organisms  will 
remove  the  agglutinin  completely  from  immune  sera,  while  closely 
related  bacteria  only  remove  that  portion  of  the  agglutinating  sub- 
stance which  is  common  to  both  organisms,  leaving  behind  the 
specific  agglutinin  which  will  then  agglutinate  the  specific  organism, 
but  not  its  closely  related  fellow;  that  is  to  say,  closely  related 
bacteria  will  react  with  the  common  or  group  agglutinin,  but  fail  to 
absorb  the  specific  agglutinin. 

Experience  has  shown  that  the  sera  of  normal  adults  frequently 
contain  agglutinin  which  will  clump  various  bacteria  and  the  potency 
of  these  "normal"  or  natural  agglutinins  may  even  be  sufficient  to 
clump  moderate  numbers  of  typhoid  bacilli  in  dilutions  as  great  as 
1  to  30.  The  sera  of  normal  nurslings  contain  only  minimal  amounts 
of  normal  agglutinins  as  a  rule,  and  the  conclusion  has  been  drawn 
that  normal  agglutinin  may  be  either: 

(a)  Group  agglutinin,  derived  from  mild  infection  with  closely 
related  organisms,  or 

(6)  True  immune  agglutinins  resulting  from  mild  or  unrecognized 
infection  with  the  specific  organism. 

No  definite  distinction  has  been  noted  between  natural  and  immune 
agglutinins;  the  latter  are  usually  present  in  sera,  however,  in  much 
greater  concentration  than  the  former. 

The  site  of  formation  of  agglutinins  in  the  body  is  not  definitely 
known,  although  lymphoid  tissues  appear  to  be  intimately  concerned, 
especially  bone-marrow  and  the  spleen.  Pryzgode3  states  that 

1  Jour.  Med.  Res.,  August,  1903,  x,  No.  1. 

2  Bordet,  Collected  Studies  in  Immunity,  1909  (translation  by  Gay). 

3  Wien.  klin.  Wchnschr.,  1913,  xxvi,  84. 


AGGLUTININS— AGGLUTINOIDS  AND  PROAGGLUTINOIDS   145 

cultures  of  spleen  tissue  in  vitro  will  form  specific  agglutinins  for 
typhoid  bacilli  if  the  virus  is  brought  into  contact  with  the  tissue 
cells.  As  a  general  rule  the  concentration  of  a  specific  agglutinin  is 
greater  in  the  blood  stream  than  in  the  tissues  of  the  body. 

Preparation  of  Specific  Agglutinating  Sera. — Specific  agglutinating 
sera  for  experimental  purposes  are  best  obtained  from  rabbits,  whose 
serum  normally  contains  no  agglutinin.  Several,  usually  three  to 
five  intravenous  injections  of  1,  2,  3  and  5  loopfuls  respectively  of 
killed  cultures  of  typhoid  bacilli  at  eight-day  intervals,  produce  pow- 
erful agglutinating  sera.  The  animal  is  bled  about  two  weeks  after 
the  last  injection.  For  large  amounts  of  agglutinating  sera  horses  or 
asses  must  be  used. 

Properties  of  Agglutinins. — Agglutinins  are  of  unknown  chemical 
composition,  but  they  may  be  separated  from  solution  by  those  pre- 
cipitants  wrhich  throw  down  globulins,  and  they  may  be  removed 
from  solution  by  absorption  in  animal  charcoal.  Toward  heat  they 
are  moderately  resistant,  usually  remaining  active  after  an  exposure 
of  twenty  minutes  to  55°  C.,  a  degree  of  heat  sufficient  to  inactivate 
complement.  Agglutinins,  therefore,  appear  to  be  quite  distinct 
from  bacteriolysins.  The  temperature  at  which  agglutinins  are  de- 
stroyed depends  upon  their  specificity,  agglutinins  for  plague  bacilli 
being  more  sensitive  than  typhoid  agglutinins.  The  reaction  of  the 
medium  also  affects  .their  stability.  Alkalis,  even  in  dilute  solution, 
rapidly  destroy  agglutinins;  acids  are  somewhat  less  harmful.  Nat- 
urally the  duration  of  exposure  to  these  various  agents  exercises  an 
important  influence  upon  their  resistance.  Agglutinins  do  not  appear 
to  pass  through  parchment  membranes,  but  it  is  stated  that  agglu- 
tinogen  will  slowly  diffuse  under  similar  conditions.  This  would  sug- 
gest that  the  agglutinin  molecule  is  larger  than  the  agglutinogen 
molecule.  Preserved  in  a  dry  state,  in  a  cool  place  away  from  light, 
agglutinins  preserve  their  properties  unimpaired  for  days.  In 
solution  and  upon  standing  agglutinins  rapidly  lose  their  property 
of  clumping  bacteria,  but  they  still  retain  their  original  ability  to  unite 
firmly  with  bacteria.  Ehrlich  designates  agglutinins  which  have 
lost  their  ability  to  cause  clumping  but  still  retain  their  combining 
power  for  agglutinogen,  agglutinoids.  In  his  terminology  they  are 
side-chains  of  the  second  order  which  have  lost  their  agglutinophore 
(ergophore)  group.  Agglutinins  acting  in  neutral  salt-free  media 
also  fail  to  cause  clumping  of  bacteria,  but  in  this  case  the  addition 
of  a  small  amount  of  NaCl  or  even  some  weak  acid  very  soon  brings 
10 


146    ANTIGENS  AND   THE.TECHNIC  OF  SERUM  REACTIONS 

about  a  typical  reaction.1  This  and  similar  observations  have 
attracted  attention  to  the  similarity  between  the  precipitation  of 
bacteria  to  which  agglutinin  is  anchored  by  neutral  salts,  and  the 
precipitation  of  finely  suspended 'clay  by  the  addition  of  neutral  salts; 
the  inference  has  been  drawn  that  the  phenomenon  of  agglutination 

one  of  physico-chemistry. 

Specificity  of  Agglutination  Reactions:  Group  Agglutinins. — The 
composition  of  the  agglutinogen — that  constituent  of  the  bacterium 
which  stimulates  agglutinin  formation — is  unknown,  but  it  appears 
to  be  complex  and  probably  not  a  single  chemical  compound.  Closely 
related  bacteria  may  possess  in  common  a  small  amount  of  agglu- 
tinogen— a  least  common  multiple,  as  it  were — which  stimulates  the 
production  of  "group  agglutinin"  that  reacts  with  related  bacteria 
more  or  less  in  proportion  to  their  content  of  the  common  antigen  or 
agglutinogen.  .  The  specific  agglutinin  produced  by  the  entire  agglu- 
tinogen content  of  an  organism  is  more  potent  and  fails  to  react  with 
related  bacteria.  Thus,  the  serum  of  an  animal  immunized  against 
B.  typhosus  may  agglutinate  that  organism  in  a  dilution  of  1  to  3000; 
B.  paratyphosus  will  be  agglutinated  in  a  dilution  of  1  to  300  by  the 
same  serum,  and  B.  coli  would  agglutinate  only  in  a  dilution  of  1  to 
50.  The  group  agglutinin  in  this  example  would  be  effective  for  B. 
paratyphosus  in  a  dilution  of  1  to  300,  but  in  greater  dilutions  it  would 
be  ineffective.  For  B.  coli  in  the  instance  cited,  the  group  agglutinin 
is  ineffective  in  dilutions  above  1  to  50. 

The  common  or  group  agglutinin  for  B.  paratyphosus  in  this  typhoid 
serum  could  be  quantitatively  removed  by  leaving  it  in  contact  with 
a  large  number  of  paratyphoid  bacilli  for  a  few  hours,  then  centrifu- 
galizing  to  remove  the  organisms.  The  residual  serum  would  contain 
only  agglutinin  specific  for  B.  typhosus.  If  B.  typhosus  were  added 
to  the  serum,  all  the  agglutinin — both  "group"  and  specific — would 
be  removed. 

As  a  general  rule,  group  agglutinins  constitute  a  minor  fraction  of 
the  total  agglutinin  and  in  practice  the  degree  of  dilution  of  the  serum 
used  in  specific  cases  is  ample  to  exclude  error.  It  occasionally  happens 
that  sera  of  low  dilution,  especially  those  rich  in  agglutinoids,  fail  to 
clump  the  specific  organism;  as  the  serum  is  diluted  more  and  more 
the  phenomena  of  clumping  become  more  and  more  marked;  finally 
a  degree  of  dilution  is  reached  beyond  which  the  serum  again  becomes 
ineffective.  The  initial  negative  agglutination  in  concentrated  serum 

1  Bordet,  Ann.  Inst.  Past.,  1899,  xiii,  225. 


AGGLUTININS— AGGLUTINOIDS  AND  PROAGGLUTINOIDS       147 

is  known  as  a  " proagglutinoid"  reaction;  it  is  attributed  by  Ehrlich 
to  the  presence  of  "  agglutinoids"  in  the  serum — side-chains  of  the 
second  order  which  have  lost  their  agglutinophore  group,  but  still 
retain  their  combining  group  (haptophore  group).  These  " agglu- 
tinoids," which  are  deteriorated  agglutinins,  have  a  greater  affinity 
for  the  agglutinogen  of  the  bacteria  than  have  the  unchanged  agglu- 
tinins, and  consequently  prevent  the  latter  from  becoming  attached 
to  the  organisms.  If  the  serum  is  diluted  a  point  is  reached  where 
the  agglutinoids  are  numerically  too  few  to  interfere  with  the  action 
of  the  agglutinins,  which  usually  far  outnumber  the  agglutinoids.  As 
the  serum  is  more  and  more  diluted  a  point  is  eventually  reached 
where  the  content  of  agglutinin  is  insufficient  to  react  with  the  bacteria. 
If,  however,  bacteria  are  cultured  in  this  dilute  serum,  they  frequently 
develop  into  long,  thread-like,  interwoven  filaments,  the  so-called 
"thread-reaction"  of  Pfaundler.  It  is  obvious  that  the  maximum 
dilution  at  which  a  serum  will  agglutinate  bacteria  depends  somewhat 
upon  the  number  of  organisms;  there  is,  in  other  words,  a  relation 
between  the  amount  of  agglutinin  in  the  serum  and  agglutinogen  in 
the  bacteria. 

Non-agglutinable  Bacteria. — Occasionally  strains  of  bacteria,  as 
B.  typhosus,  freshly  isolated  from  the  body,  may  not  agglutinate 
with  the  specific  serum.  This  resistance  to  agglutination  is  supposed 
to  result  from  some  unknown  change  in  the  agglutinogen  of  the  bac- 
terium during  its  development  in  the  body.  A  similar  loss  of  agglu- 
tinability  may  be  experimentally  brought  about  by  growing  the 
bacteria  in  gradually  increasing  concentrations  of  specific  agglutinat- 
ing serum  outside  the  body.  This  inagglutinability  is  usually  lost 
after  a  few  days'  development  on  artificial  media;  the  organisms  will 
then  clump  in  a  characteristic  manner  in  a  serum  that  originally  was 
ineffective. 

The  Reaction  of  Agglutination. — The  practical  value  of  the  reaction 
of  agglutination  depends  upon  the  visible  clumping  or  agglomeration 
of  a  suspension  of  bacteria  in  a  fluid  medium  containing  some  neutral 
salt,  when  a  relatively  small  amount  of  immune  serum  specific  for 
the  organism  is  added  to  it.  The  reaction  may  be  expressed  thus: 

Organism  (Agglutinogen)  +  Specific  Serum  (Specific  Agglutinin) 
=  Agglutination. 

If  a  specific  organism  is  added  to  an  appropriate  dilution  of  unknown 
serum  with  proper  precautions,  and  characteristic  clumping  takes 
place,  or  if  a  known  specific  serum  is  added  with  suitable  precautions 


148  ANTIGENS  AND  THE  TECHNIC  OF  SERUM  REACTIONS 

to  a  suspension  of  an  unknown  organism,  and  characteristic  clumping 
takes  place,  a  specific  diagnosis  of  the  serum  or  of  the  organism  can 
be  arrived  at.  In  the  first  instance,  a  diagnosis  of  disease  may  be 
made;  in  the  second  instance  the  identity  of  an  organism  may  be 
established.  The  laboratory  diagnosis  of  typhoid  and  paratyphoid 
fever,  of  the  various  types  of  bacillary  dysentery  and  of  other  bacterial 
infections  is  frequently  made  by  testing  the  serum  of  the  patient 
for  agglutination  with  a  known  culture  of  the  organism.1  The  labora- 
tory identification  of  specific  bacteria,  conversely,  is  frequently  estab- 
lished or  corroborated  through  their  agglutination  with  known  specific 
agglutinating  sera. 

The  reaction  of  agglutination  may  be  made  either  microscopically 
or  macroscopically. 

1.  Microscopic  Method. — A  drop  of  serum  from  a  patient,  diluted  to 
the  proper  degree,  is  mixed  with  an  equal  amount  of  a  broth  culture 
of  the  desired  organism  on  a  clear  cover-glass,2  and  then  suspended 
over  the  cavity  of  a  hollow  ground  slide,  ringed  with  vaseline  to  pre- 
vent evaporation,  and  examined  under  the  microscope.    Motile  bac- 
teria, as  for  instance  B.  typhosus,  soon  lose  their  motility  (immo- 
bilization) and  gradually  collect  in  small  groups  which  tend  to  coalesce 
into  larger  and  larger  clumps,  leaving  the  field  between  them  practically 
free  from  organisms.    The  bacteria  are  not  necessarily  killed  by  agglu- 
tination.    The   reaction   ordinarily   is   complete   within  two   hours. 
Killed  cultures  of  bacteria  may  be  used  in  place  of  living  cultures 
but  the  reaction  is  usually  less  clear-cut. 

The  advantage  of  the  microscopic  method  lies  chiefly  in  the  small 
amount  of  serum  required  to  perform  the  test.  One  of  its  chief  disad- 
vantages lies  in  the  relative  inaccuracy  of  the  dilution  of  the  serum. 
(See  chapter  on  B.  typhosus  for  full  discussion  of  technic.) 

2.  Macroscopic   Method. — Various   dilutions   of   serum,    accurately 
measured  by  volumetric  pipettes,  are  brought  into  small,  sterile  test- 
tubes,  together  with  suspensions  or  broth  cultures  of  the  bacteria. 
Agglutination  is  manifested  by  the  gradual  accumulation  of  a  floccu- 
lent  sediment  of  bacteria,  leaving  the  supernatant  liquid  perfectly 
clear.     Control  tubes  without  serum  remain  uniformly  clouded. 

The   part  played  by   agglutinins   in   immunity   is   unknown;  the 

1  The  technic  and  precautions  to  be  observed  are  discussed  individually  in  the  chap- 
ters upon  specific  pathogenic  bacteria. 

2  For  a  majority  of  bacteria,  eighteen-hour  cultures  in  0.1  per  cent,  dextrose  broth 
are  particularly  advantageous.     Cultures  grown  in  plain  broth  are  usually  much  less 
actively  motile  and  agglutinate  less  readily. 


PRECIPITINS—PRECIPITINOIDS  149 

concentrations  of  agglutinins  in  immune  sera,  as  measured  by  present- 
day  methods,  throws  no  light  upon  the  degree  of  immunity  or  the 
prognosis.  Very  severe  typhoid  infections,  for  example,  may  show 
little  agglutinin  in  their  sera,  and  mild  cases  may  exhibit  sera  com- 
paratively rich  in  agglutinin  content.  Their  chief  value  at  the  present 
time  lies  in  their  relation  to  the  diagnosis  of  disease. 

PRECIPITINS.   PRECIPITINOIDS. 

In  the  preceding  section  it  was  shown  that  the  sera  of  animals 
immunized  with  various  bacteria  contained  substances — agglutinins 
—which  agglutinated  the  specific  organisms.  Kraus1  showed  that 
these  immune  sera  would  cause  a  precipitate  when  they  were  added 
to  clear  filtrates  of  the  specific  organisms.  During  the  process  of 
immunization,  therefore,  specific  antibodies,  termed  precipitins,  are 
formed,  which  react  with  the  specific  soluble  antigen,  precipitinogen, 
in  germ-free  filtrate  of  broth  cultures  of  the  specific  organisms,  to 
form  a  precipitate.  Later  investigations  have  shown  that  any  soluble 
protein,  as  egg-albumen,  injected  into  experimental  animals  may 
stimulate  the  production  of  specific  precipitins  which  will  cause  a 
precipitation  in  clear  solutions  of  the  homologous  protein.  These 
reactions  have  a  marked  specificity:  The  sera  of  animals  immunized 
against  casein  of  cows'  milk,  for  example,  will  cause  precipitation  in 
clear  solutions  of  this  protein,  but  will  fail  to  cause  precipitation  in 
solutions  of  casein  from  human  milk.  The  sera  of  closely-related  ani- 
mals may  contain  small  amounts  of  "group"  precipitins,  and  biological 
relationships  have  been  established,  based  upon  the  community  of 
these  antibodies.  Thus,  the  sera  of  certain  anthropoid  apes2  are 
said  to  be  precipitated  by  the  sera  of  animals  immunized  to  the  serum 
of  man;  sera  from  the  lower- monkeys  fail  to  react  with  the  human 
serum.  From  these  observations  the  inference  has  been  drawn  that 
these  anthropoid  apes  are  more  closely  related  to  man  than  are  the 
lower  monkeys.3 

Precipitins  closely  resemble  agglutinins  in  their  method  of  formation, 
their  resistance  to  physical  agents  and  their  reactions.  Like  the 
agglutinins,  they  possess  both  a  thermostabile  haptophore  or  combining 
group  and  a  thermolabile  ergophore  group.  The  precipitinophore 

1  Wien.  klin.  Wchnschr.,  1897,  736. 

2  Griinbaum,  Lancet,  January,  1902. 

3  See  Nuttall,  Jour.  Hyg.,  1901,  i,  No.  3;  Proc.  Royal  Acad.,  November,  1901,  Ixix; 
Proceedings  Cambridge  Philosophical  Society,  January,  1902;  Brit.  Med.  Jour.,  April, 
1902,  i,  for  full  details. 


150  ANTIGENS  AND  THE  TECHNIC  OF  SERUM  REACTIONS 

group  is  very  labile  and  readily  becomes  non-functionating,  but  the 
combining  group  is  relatively  stabile.  A  precipitin  which  has  lost 
its  ergophore  group  is  called  a  precipitinoid. 

The  precipitate  formed  by  a  specific  serum  acting  upon  a  clear 
solution  of  the  antigen  (precipitinogen)  probably  is  derived  from  the 
serum,  because  very  dilute  solutions  of  the  immunizing  protein  will 
throw  down  a  relatively  bulky  precipitate,  far  too  great  in  amount 
to  come  from  the  antigen  in  the  dilution  used.1 

Precipitins  have  been  extensively  studied  in  their  relation  to  cer- 
tain aspects  of  Forensic  Medicine,  but  they  have  little  practical  value 
in  the  laboratory  diagnosis  of  bacterial  disease.  They  are  found  in 
sera  under  the  same  conditions  as  agglutinins,  but  the  technic  for  their 
demonstration  is  more  involved  than  that  for  agglutinins.  Their 
relation  to  immunity  is  unknown,  but  probably  similar  to  that  of 
agglutinins. 

LYSINS. 

Mention  has  been  made  (see  preceding  section)  of  the  bactericidal 
power  of  fresh  blood  serum  of  a  normal  animal  and  man.  This  impor- 
tant discovery,  that  normal  sera  contain  substances  that  will  destroy 
moderate  numbers  of  bacteria,  was  made  by  Nuttall,2  who  also 
observed  that  there  was  a  limit  to  this  destructive  activity  and  that 
this  property  was  lost  upon  standing,  or  rapidly  destroyed  by  an 
exposure  of  the  serum  to  55°  C.  for  half  an  hour.  Buchner3  corrobo- 
rated and  extended  these  observations  and  designated  the .  unknown 
stabile  component  "alexin."  Pfeiffer4  then  showed  that  the  destruc- 
tive action  of  normal  sera  could  be  increased  many  fold  above  its 
original  level  for  a  specific  organism  if  that  organism  were  repeatedly 
injected  into  an  animal  in  sublethal,  but  gradually  increased  doses. 
The  serum  of  such  an  animal  would  still  destroy  only  moderate  num- 
bers of  heterologous  bacteria,  but  relatively  great  numbers  of  the 
homologous  bacteria.  This  observation  opened  the  way  for  the  highly 
important  study  of  active  acquired  immunity  against  bacteria. 
Pfeiffer  observed  that  heating  immune  sera  to  50°  to  56°  C.  for  half 
an  hour  destroyed  their  bactericidal  properties,  precisely  as  Nuttall 
had  found  that  natural,  non-specific  bactericidal  properties  were 
destroyed  under  similar  conditions.  Bordet5  then  discovered  that  the 

Welsh  and  Chapman,  Ztschr.  f.  Immunitasforsch.,  1911,  ix,  517. 

Ztschr.  f.  Hyg.,  1888,  iv,  353. 

Cent.  f.  Bakt.,  1889,  v,  817;  vi,  1,  561. 

Ztschr.  f.  Hyg.,  1894,  xviii,  1 ;  1895,  xix,  75-100. 

Ann.  Inst.  Past.,  1895. 


LYSINS  151 

addition  of  a  small  amount  of  unheated  blood  serum  from  a  non- 
immune  animal  would  "reactivate"  the  heated  inactive  immune  serum 
and  restore  its  bactericidal  power  to  its  original  level.  These  experi- 
ments collectively  demonstrated  clearly  that : 

1.  Normal  sera  had  an  inherent  but  limited  destructive  action  upon 
a  variety  of  bacteria. 

2.  That  this  destructive  or  bactericidal  action  could  be  greatly 
increased  for  specific  organisms  through  repeated  injections  of  sub- 
lethal  doses  of  them.1 

3.  That  both  normal  and  immune  sera  lost  their  bactericidal  prop- 
erties by  heating  them  to  55°  C.  for  half  an  hour. 

4.  That  immune  sera  would  regain  their  specific  bactericidal  power 
if  a  small  amount  of  fresh  normal  blood  serum  of  a  non-immune 
animal  were  added  to  them.2 

Bordet3  showed  similarly  that  the  red  blood  cells  of  an  alien  animal 
were  also  destroyed  to  a  limited  degree  by  the  serum  of  a  normal 
'animal,  but  that  the  destruction  could  be  greatly  increased  for  specific 
erythrocytes  if  they  were  repeatedly  injected  into  an  experimental 
animal.  The  blood  serum  becomes  specifically  hemolytic.  Here 
again  Bordet4  found  that  heating  an  immune  serum  to  55°  C.  for 
thirty  minutes  destroyed  its  activity,  but  that  a  small  amount  of 
fresh  serum  from  a  non-immune  animal  (whose  serum  per  se  would 
not  dissolve  the  homologous  cells)  would  reactivate  the  serum.  Thus, 
both  specific  bacteriolytic  sera  and  specific  hemolytic  sera  must  con- 
tain two  distinct  components — a  thermostabile  component  resisting 
an  exposure  to  55°  C.  for  half  an  hour  and  contained  only  in  the 
immune  serum,  and  a  thermolabile  component  destroyed  or  inacti- 
vated at  55°  C.,  which  is  present  both  in  active  immune  bacteriolytic 
and  hemolytic  sera,  and  also  in  normal  sera.  To  the  thermolabile 
substance  present  in  unheated  normal  and  immune  sera,  Bordet  gave 
the  name  "alexin;"  to  the  thermostabile  specific  substance  in  immune 
sera  he  gave  the  name  "substance  sensibilitrice."  He  regarded  the 
"substance  sensibilitrice"  as  a  sensitizer  or  mordant  which  made 
bacteria  or  blood  cells  vulnerable  to  the  ferment-like  or  digestive 
action  of  the  "alexin." 

Ehrlich  and  Morgenroth5  studied  the  phenomena  of  hemolysis  in 

1  Presumably  leaving  the  original  non-specific  bactericidal  power  at  its  initial  level 
for  all  except  the  specific  organism,  and  possibly  for  closely  related  forms. 

2  Moxter,  Cent.  f.  Bakt.,  1899,  xxvi,  344. 

3  Loc.  cit. 

4  Ann.  Inst.  Past.,  1898,  xii,  No.  10. 

6  Berl.  klin.  Wchnschr.,  1899,  No.  1  and  22.    See  also  Collected  Studies  on  Immunity, 
Ehrlich,  translated  by  Bolduan,  1910. 


152   ANTIGENS  AND   THE   TECHNIC  OF  SERUM  REACTIONS 

great  detail  and  demonstrated  by  very  careful  and  ingenious  experi- 
ments that  the  phenomena  observed  by  Bordet  were  fundamentally 
correct.  They  showed : 

1.  That  inactivated  specific  hemolytic  serum  (heated  to  55°  C.) 
was  absorbed  by  the  homologous  red  blood  cells,  and  that  these  "  sen- 
sitized "  cells,  separated  from  the  serum  after  a  few  hours  and  washed 
carefully,  were  readily  hemolyzed  when  resuspended  in  salt  solution 
to  which  was  added  a  small  amount  of  fresh,  unheated,  normal  guinea- 
pig  serum. 

2.  The  supernatant  residual  fluid  from  which  the  red  blood  cells 
had  removed  all  the  immune  body  was  incapable  of  causing  hemolysis 
of  the  homologous  red  blood  cells  when  fresh  normal  serum  was  added 
to  it.    The  erythrocytes,  in  other  words,  quantitatively  removed  the 
thermostabile  "substance  sensibilitrice"  from  solution. 

3.  If  normal  sera  were  allowed  to  remain  in  contact  with  the  same 
red  cells  for  an  equal  length  of  time,  and  these  red  cells  were  then 
removed  by  centrifugalization  and  resuspended  in  salt  solution  con- 
taining normal  fresh  serum,  no  hemolysis  took  place,  leading  to  the 
conclusion  that  the  thermolabile  substance  (alexin  of  Bordet)  is  not 
removed  from  solution  by  erythrocytes.     Apparently  alexin  is  not 
bound  or  anchored  directly  to  the  red  cells. 

4.  Finally,  it  was  shown  that  inactivated  immune  serum,  red  blood 
cells  and  fresh  normal  serum  could  be  maintained  at  0°  C.  without 
apparent  hemolysis.    At  37°  C.  the  same  solution  soon  exhibited  com- 
plete hemolysis.    Thus,  at  the  lower  temperature,  the  normal  serum 
failed  to  cause  hemolysis.    If  the  mixture  maintained  at  0°  C.  were 
centrnugalized,  however,  after  some  hours,  and  the  red  blood  cells 
washed    thoroughly    and    resuspended    in    salt    solution,    hemolysis 
promptly  occurred  when  a  small  amount  of  normal  serum  was  added 
to  the  suspension,  thus  showing  clearly  that  the  inactivated  immune 
serum  was  bound  or  anchored  by  the  red  blood  cells  at  0°  C.,  even 
though  activation  did  not  take  place. 

Ehrlich  substituted  the  term  "  amboceptor"  for  Bordet's  term 
"substance  sensibilitrice"  and  complement  for  the  term  "alexin," 
and  conceived  that  the  immune  body — amboceptor — consisted  essen- 
tially of  two  combining  or  haptophore  groups — one  the  cytophilic 
group,  possessing  a  specific  combining  power  for  the  specific  cell 
(bacterium  or  erythrocyte),  the  other,  complementophilic  group, 
combining  with  the  non-specific  complement.  According  to  this 
theory  the  union  of  complement  to  specific  cell  takes  place  through  the 


LYSINS  153 

amboceptor;  Bordet  maintains  that  neither  the  specific  cell  (antigen) 
of  itself  nor  the  substance  sensibilitrice  (amboceptor)  of  itself  unites 
with  alexin  (complement).  When  both  are  simultaneously  present, 
however,  alexin  is  absorbed.  In  other  words,  amboceptors  as  such  do 
not  exist,  according  to  this  view,  and  consequently  complement  can- 
not be  bound  to  the  specific  cell  by  a  complementophile  (haptophore) 
group. 

Multiplicity  of  Amboceptors  and  Complement. — The  researches  of 
Nuttall  and  Buchner  and  of  Moxter1  have  shown  that  fresh  normal 
serum  possesses  definite  but  limited  bactericidal  powers,  apparently 
not  specific  (for  a  variety  of  bacteria  may  be  destroyed)  which  are 
destroyed  by  an  exposure  of  thirty  minutes  to  55°  C.  Furthermore, 
the  "inactivated"  serum  appears  to  regain  its  original  bactericidal 
value  for  various  organisms  when  it  is  mixed  with  a  relatively  small 
amount  of  normal  serum.  In  other  words,  normal  serum  and  specific 
immune  serum  (unheated)  alike  appear  to  depend  upon  thermostabile 
amboceptor  and  thermolabile  complement  for  their  bacteriolytic  and 
hemolytic  activities.  They  differ  in  the  highly  specific  potency  of 
the  immune  serum  for  its  homologous  cell.  Ehrlich  and  Morgenroth2 
believe  that  the  normal  or  natural  cytolytic  activities  of  sera  depend 
upon  a  multiplicity  of  specific  amboceptors,  each  for  its  specific  red 
blood  cell  or  other  cell,  and  Pfeiffer3  has  made  similar  observations 
for  the  normal  bactericidal  powers  of  blood.  Ehrlich  and  Morgen- 
roth have  attempted  to  demonstrate  a  multiplicity  of  complements  in 
normal  sera  also;  heated  normal  sera  injected  into  normal  animals 
are  claimed  by  the  Ehrlich  school  to  give  rise  to  anticomplementoids, 
the  supposition  being  that  the  heat  has  destroyed  the  ergophore 
group  of  complement  but  not  its  combining  group,  giving  rise  to  a 
"complementoid,"  precisely  as  a  toxin  which  has  lost  its  toxophore 
group  becomes  a  toxoid.  There  appears  to  be  no  theoretical  limit 
to  the  anti-  and  anti-antibodies  which  may  thus  be  produced  by 
various  increasingly  complicated  investigations.  Bordet  and  Gay4 
deny  the  multiplicity  of  complement. 

Fixation  of  Complement. — Bordet  and  Gengou,5  in  a  series  of  experi- 
ments, brought  forth  experimental  evidence  of  the  unity  of  com- 
plement and,  incidentally,  developed  a  method  of  investigation  now 

1  Loc.  cit.  2  LOC>  eft. 

3  Harben  Lecture,  Jour.  Royal  Inst.  Public  Health,  1909,  xvii,  385. 

4  Collected  Studies  in  Immunity  by  Bordet  and  his  associates  (translated  by  Gay, 
1909). 

8  Ann.  Inst.  Past.,  1901,  xv. 


154   ANTIGENS  AND   THE  TECHNIC  OF  SERUM  REACTIONS 

extensively  utilized  to  demonstrate  the  presence  of  various  specific 
immune  antibodies.  If  a  specific  immune  body  (as  for  example, 
the  serum  of  an  animal  immunized  to  typhoid  bacilli)  .is  heated  to 
55°  C.  for  half  an  hour,  then  added  to  a  suspension  of  typhoid  bacilli 
together  with  normal  unheated  serum,  a  union  between  the  bacilli, 
the  specific  antibody  of  the  serum  (amboceptor,  substance  sensibili- 
trice)  and  the  complement  (alexin)  will  take  place.  If  the  proportions 
of  the  three  reactive  bodies  are  correct,  all  the  complement  or  alexin 
will  be  bound,  provided  the  mixture  is  incubated  a  few  hours  at  37° 
C.  If,  now,  red  blood  cells  and  inactivated  immune  serum  specific 
for  the  red  blood  cells  are  added  to  the  mixture  of  bacteria,  immune 
body  and  complement,  no  hemolysis  should  be  noticed,  because  the 
complement  is  quantitatively  anchored  to  the  bacteria-immune  serum 
complex.  If,  on  the  other  hand,  the  inactivated  immune  serum  added 
to  the  suspension  of  typhoid  bacilli  be  not  typhoid  immune  serum, 
complement  will  not  be  bound  to  the  bacteria,  for  the  specific  ambo- 
ceptor or  substance  sensibilitrice  will  not  be  present.  The  complement 
or  alexin,  therefore,  is  not  anchored  to  the  bacteria,  and  it  is  free  to 
act  when  the  red  blood  cells  and  their  specific  inactivated  serum  are 
added  to  the  mixture  of  bacteria  and  serum.  Under  this  condition 
hemolysis  occurs,  because  the  red  blood  cells,  inactive  immune  body 
and  complement  unite.  The  production  of  hemolysis  being  visible, 
it  acts  as  an  indicator  in  such  instances.  Wassermann  and  his  asso- 
ciates have  utilized  this  method  of  "fixation  of  complement"  for  the 
serologic  diagnosis  of  syphilis,  and  gradually  a  relatively  large  number 
of  diagnoses  of  clinical  importance  have  been  developed  along  the 
same  lines. 

The  Determination  of  Specific  Antibodies  by  the  Method  of  Complement 
Fixation. — Principle  Invoked. — When  an  antigen  (bacteria,  erythro- 
cytes,  tissue  cells,  protein,  or  other  substance  which  stimulates  specific 
antibody  formation)  is  mixed  intimately  with  its  specific  inactivated 
immune  serum  and  fresh  normal  complement  a  firm  union  of  the 
three  components  takes  place.1  Jhe  result  of  this  union  is  an  injury 
or  destruction  of  the  antigen,  ^f  the  antigen  be  bacterial  cells  or 
tissue  cells  there  is  usually  no  visible  change  in  the  gross  appearance 
of  the  mixture,  and  cultural  or  chemical  investigation  must  be  relied 
upon  to  demonstrate  the  lytic  process.  Erythrocytes,  on  the  other 
hand,  undergo  changes  in  the  presence  of  inactivated  specific  immune 
serum  and  complement  which  result  in  the  liberation  of  hemoglobin, 

1  Bordet  and  Gengou,  Ann.  Inst.  Past.,  1901,  xv,  290. 


LYSIN&  155 

which  colors  the  solution  deep  red.  This  change  is  clearly  visible 
and  requires  no  additional  procedure  for  its  demonstration;  the 
liberation  of  hemoglobin  is  in  itself  an  indicator  of  the  reaction  which 
has  taken  place. 

The  relation  between  antigen,  immune  serum,  and  complement 
is  quantitative;  consequently,  if  the  respective  amounts  of  the  three 
components  are  correctly  proportioned,  no  free  unattached  comple- 
ment will  be  present  in  a  mixture  of  them  after  an  appropriate  incuba- 
tion at  body  temperature  is  practiced  to  allow  of  their  union.  Usually 
an  hour  at  37°  C.  suffices  for  this  union  to  take  place  quantitatively. 

These  very  important  observations  of  Bordet  and  Gengou  have 
led  to  the  development  of  a  technic  for  the  diagnosis  of  infection,  and 
the  identification  of  antigens  by  the  method  of  complement  fixation. 

The  underlying  principles  of  the  reaction  of  complement  fixation 
are  three: 

(a)  The  union  of  specific  inactivated  immune  serum  and  homologous 
antigen. 

(b)  The  quantitative  activation  of  the  antigen — inactivated  specific 
immune  serum  complex  by  non-specific  complement;  and 

(c)  The  visible  hemolysis  that  results  from  the  activation  of  an 
erythrocyte — inactivated   specific   immune   serum  complex  by  non- 
specific complement. 

The  general  plan  of  procedure  is  to  incubate  an  antigen  (as  bacterial 
cells)  and  inactivated  serum  and  complement  in  proper  proportions 
for  an  hour,  to  permit  the  three  components  to  unite.  A  mixture  of 
erythrocytes  and  specific  inactivated  hemolytic  serum  is  now  added. 
If  the  reactive  substances  are  properly  proportioned  and  the  inac- 
tivated serum  first  added  is  specific  for  the  antigen  (bacteria),  no 
hemolysis  will  occur  when  the  hemolytic  system  is  added,  because 
all  the  complement  present  is  bound  by  the  bacteria-immune  serum 
complex.  On  the  contrary,  if  the  inactivated  serum  is  not  specific 
for  the  bacterial  antigen,  no  union  between  the  two  will  take  place, 
complement  will  not  be  bound,  and  it  is  free  in  the  mixture.  It  will 
activate  the  erythrocyte-inactivated  immune  serum  complex,  and 
hemolysis  will  occur. 

It  will  be  seen  that  the  hemolytic  system  is  added  as  an  indicator; 
an  absence  of  hemolysis  shows  a  union  of  bacterial  antigen,  inactive 
specific  bacterial  immune  serum  and  complement.  Hemolysis  shows 
that  the  union  has  not  been  formed,  the  complement  was  free  in  the 
mixture  and  it  united  with  the  hemolytic  system,  causing  hemolysis 


156     ANTIGENS  AND  THE  TECHNIC  OF  SERUM  REACTIONS 

in  the  erythrocyte  antigen  through  the  specific  amboceptor  or 
hemolysin. 

The  method  of  complement  fixation  may  be  employed  to  examine 
sera  for  specific  antibodies,  using  a  known  antigen,  or  to  test  suspected 
antigens  with  sera  containing  specific  antibodies.  The  most  practical 
application  of  the  method  in  medicine  is  the  serum  diagnosis  of  syphilis, 
glanders,  and  other  bacterial  infections.  t 

The  Technic  of  Complement  Fixation. — The  technic  of  complement 
fixation  is  simple  in  principle,  but  it  requires  the  most  scrupulous 
attention  to  details.  All  glassware  must  be  neutral  in  reaction, 
chemically  clean,  and  bacteriologically  sterile.  Physiological  salt 
solution  (0.85  to  0.90  per  cent.  C.P.  NaCl  in  neutral  distilled  water) 
used  for  washing  red  blood  cells  and  for  dilutions  should  be  sterile 
and  stored  in  clean  containers. 

The  Wassermann  Serum  Diagnosis  of  Syphilis. — Five  elements  enter 
into  the  Wassermann  test  for  syphilis:  the  antigen,  suspected  syphilitic 
serum,  complement,  and  a  hemolytic  system  consisting  of  red  blood 
cells  and  specific  immune  hemolytic  serum  (hemolysin). 

Preparation  and  Standardization  of  Antigen. — The  antigen  originally 
employed  by  Wassermann  and  his  collaborators  was  an  aqueous 
extract  of  syphilitic  tissue  which  was  prepared  by  suspending  one  part 
by  weight  of  finely  comminuted  liver  of  a  syphilitic  fetus1  in  five  parts 
of  physiological  salt  solution  containing  0.5  per  cent,  phenol  as  a 
preservative.  After  several  days'  violent  agitation  in  the  dark  it  is 
strained  through  several  layers  of  cheesecloth  to  remove  coarser  par- 
ticles and  stored  in  amber  bottles  in  the  refrigerator.  Sedimentation 
takes  place  until  a  brownish,  slightly  opalescent  fluid  remains,  which 
is  the  luetic  antigen. 

Later  work2  showed  that  alcoholic  extracts  of  luetic  liver  were  more 
stable  than  watery  extracts.  The  specific  reacting  component,  accord- 
ing to  Forges  and  Meier,  is  lipoidal  in  nature,  and  in  this  sense  it  is 
not  biologically  specific;  The  fixation  of  complement  appears  to 
depend  upon  a  substance  in  the  antigen,  lipoidal  in  nature,  which 
effects  a  union  of  antigen,  immune  body  and  complement.  Citron 
has  proposed  the  term  "lues  reagine"  for  this  substance.  Alcoholic 
extracts  of  syphilitic  liver  are  prepared  by  shaking  finely  comminuted 
liver  with  ten  times  the  weight  of  absolute  alcohol  for  a  few  days, 

1  The  tissue  is  examined  for  the  specific  organism;  if  Treponemata  are  abundant  it 
is  converted  into  antigen,  otherwise  it  is  discarded. 

2  Especially  by  Forges  and  Meier,  Berl.  klin.  Wchnschr.,  1908,  No.  15. 


LYSINS  157 

then  digesting  the  mixture  at  37°  C.  for  a  week.  The  extract  is 
filtered  through  filter  paper  and  placed  in  the  refrigerator. 

Alcoholic  extracts  of  normal  organs,  prepared  in  the  same  manner 
as  luetic  livers,  have  been  found  to  be  quite  as  good  as  alcoholic 
extracts  of  syphilitic  livers  for  the  diagnosis  of  syphilis.  In  practice 
heart-muscle  of  normal  guinea-pigs,  freed  from  all  fat,  is  used. 

Noguchi's  Acetone-Insoluble  Lipoidal  Antigen.1 — Noguchi  and  others 
have  shown  that  .alcoholic  extracts  of  organs  may,  and  frequently 
do,  contain  sufficient  amounts  of  neutral  fats,  or  their  hydrolytic 
cleavage  products,  to  make  the  antigen  hemolytic  or  anticomplemen- 
tary.  These  substances  are  for  the  most  part  soluble  in  acetone,  while 
the  antigenic  fraction  is  insoluble  in  acetone.  One  part  of  fat-free 
heart  muscle  or  liver  from  a  guinea-pig  is  cut  into  very  fine  pieces, 
mixed  with  ten  parts  of  absolute  alcohol,  and  extracted  in  the  incu- 
bator at  37°  C.  for  a  week  or  ten  days,  being  thoroughly  shaken  every 
day.  The  soluble  substances  are  freed  from  the  fragments  of  tissue 
by  filtration  through  fat-free  filter  paper,  and  rapidly  evaporated  to 
dry  ness.2  Sufficient  ether  is  then  added  to  take  up  the  brownish 
residue,  and  it  is  then  allowed  to  stand  until  a  clear,  slightly  colored, 
ethereal  solution  is  obtained,  free  from  suspended  material.  The 
ethereal  solution  is  concentrated  by  evaporation  to  a  point  where 
separation  of  a  sediment  begins,  then  it  is  poured  into  several  volumes 
(usually  ten)  of  pure  acetone.  A  voluminous  precipitate  forms  at 
once,  and  settles  out  as  a  tenacious  gummy  mass.  This  is  retained, 
under  acetone,  as  the  antigen.  The  acetone-soluble  solution  is  dis- 
carded. The  antigen  thus  prepared  consists  largely  of  lecithins  and 
related  substances.  It  keeps  well  and  appears  to  be  very  sensitive 
and  reliable.  From  0.2  to  0.3  gram  are  dissolved  in  a  mixture  of  1 
c.c.  of  ether  (free  from  alcohol  and  having  a  neutral  reaction)  and 
10  c.c.  of  neutral  absolute  methyl  alcohol.  This  solution  is  kept 
in  an  amber  bottle  in  the  refrigerator  as  a  stock  antigen.  One  cubic 
centimeter  of  this  stock  antigen  is  added  to  19  c.c.  of  physiological 
salt  solution;  this  is  the  antigen  used  for  the  test. 

Before  making  a  test  it  is  necessary  to  standardize  the  antigen. 
It  is  essential  to  know  the  anticomplementary  titer,  that  is,  that 
maximum  amount  of  antigen  which  will  inhibit  hemolysis  in  the 
presence  of  syphilitic  serum,  but  which  will  not  inhibit  hemolysis 

1  Noguchi,  Serum  Diagnosis  of  Syphilis. 

2  Best  by  exposing  the  nitrate  in  a  broad  shallow  dish  to  an  air  current  from  an 
electric  fan. 


158    ANTIGENS  AND   THE   TECHNIC  OF  SERUM  REACTIONS 


when  non-syphilitic  serum  is  used.  In  addition,  the  following  deter- 
minations are  sometimes  desirable. 

The  hemolytic  titer,  that  amount  of  antigen  which  will  of  itself 
cause  lysis  of  red  blood  cells,  and  the  antigenic  titer,  the  amount  of 
complement  it  will  absorb  or  "fix"  in  the  presence  of  a  definite  amount 
of  specific  syphilitic  serum. 

The  anticomplementary  titration  is  made  by  mixing  graded  amounts 
of  antigen  and  a  constant  amount  of  complement  (0.1  c.c.  of  a  10  per 
cent,  solution1)  with  constant  amounts  (0.1  c.c.)  of  known  syphilitic 
serum  and  normal  serum,  both  inactivated. 

The  various  mixtures  are  incubated  in  a  water-bath  at  37°  C.  for 
an  hour,  then  0.2  c.c.  of  red  blood  cell  suspension  and  inactivated 
hemolytic  serum  are  added  and  again  incubated  in  the  water-bath  at 
37°  C.  The  maximum  amount  of  antigen  which  will  give  complete 
inhibition  of  hemolysis  with  syphilitic  serum  and  no  inhibition  of 
hemolysis  in  the  non-syphilitic  serum  is  regarded  as  the  unit. 

EXAMPLE  OF  AN  ANTICOMPLEMENTARY  TITRATION  OF  ANTIGEN. 


Normal 

Comple- 

Hemolytic 

Tube. 

Antigen. 

serum 
Inactive, 

ment,  10 
per  cent. 

Red  blood 
cells,  c.c. 

serum 
inactivated 

Result. 

c.c. 

c.c. 

units. 

1 

0.2 

0.1 

0.10 

d'0 

1.0 

1.5 

1 

Complete  hemolysis. 

2 

0.4 

0.1 

0.10 

0         * 

1.0 

1.5 

d 

"                " 

3 

0.6 

0.1 

0.10 

1.0 

1.5 

o 

"                " 

4 

0.8 

0.1 

0.10 

r§ 

1.0 

1.5 

CO    3 

"                " 

5 

1.0 

0.1 

0.10 

•5  ^ 

1.0 

1.5 

"2  £ 

Partial  inhibition. 

6 

1.5 

0.1 

0.10 

J  g 

1.0 

1.5 

J  § 

Marked  inhibition. 

7 

2.0 

0.1 

0.10 

2% 

1.0 

1.5 

c3 

Complete  inhibition. 

82 



0.1 

0.10 

os  y> 

1.0 

1.5 

-2 

03 

Complete  hemolysis. 

93 





0.10 

^    0 

1.0 

1.5 

£ 

Complete  hemolysis. 

Tube  5,  containing  1.0  c.c.  antigen,  shows  beginning  inhibition  of 
hemolysis.  This  is  regarded  as  the  anticomplementary  titer  of  the 
antigen. 

As  a  general  rule,  the  hemolytic  titer  is  higher  than  the  anti- 
complementary  titer.  The  test  is  readily  made,  if  desired,  by  using 
the  same  amounts  of  antigen  mixed  with  1  c.c.  of  red  blood  cell 
suspension  and  sufficient  salt  solution  to  bring  the  volume  to  4  c.c. 

It  is  customary  to  use  one-fourth  the  anticomplementary  titer  as 
the  standard  amount  of  antigen  to  be  used  in  the  actual  test.  In  the 

1  Prepared  by  adding  fresh  normal  guinea-pig  serum  to  physiological  salt  solution 
in  the  proportion  of  one  part  serum  to  nine  parts  salt. 

2  Serum  control.  3  Hemolytic  control. 


LYSINS  159 

example  cited,  1.0  c.c.  of  the  antigen  was  found  to  be  anticomple- 
mentary,  consequently  0.25  to  0.3  c.c.  would  be  the  proper  amount 
of  antigen  to  employ  in  the  test. 

Complement. — Fresh  guinea-pig  serum  is  the  usual  source  of  com- 
plement for  fixation  reactions.  The  animal  should  be  healthy  and 
not  previously  injected  with  protein  of  any  nature.  The  serum  of 
pregnant  pigs  is  not  trustworthy.  Blood  may  be  obtained  directly 
from  the  heart  of  the  living  animal  by  aspiration  through  a  hypo- 
dermic needle,  from  a  severed  carotid  artery,  or,  more  expeditiously 
by  cutting  the  throat  of  the  animal,  avoiding  the  esophagus,  and 
collecting  the  blood  in  sterile  Petri  dishes.  The  freshly  drawn  blood 
is  allowed  to  stand  for  a  few  hours  at  a  low  temperature  and  the  serum 
is  pipetted  off.  Complement  must  be  kept  cold  (below  16°  C.)  and  in 
the  dark.  It  must  be  used  fresh,  for  it  deteriorates  rapidly.  In  a 
frozen  condition,  however,  it  will  remain  active  for  two  or  three 
weeks.  Both  the  "activating"  and  combining  properties  of  normal 
fresh  guinea-pig  serum  are  sufficiently  constant  for  the  reaction  of 
complement  fixation. 

Hemolytic  System. — (a)  Hemolytic  Serum  (Hemolysiri). — Hemolytic 
serum  is  obtained  from  rabbits  which  have  been  injected  with  2  c.c., 
4  c.c.,  and  finally  6  c.c.  of  a  50  per  cent,  solution  of  washed  sheep 
red  blood  cells1  at  intervals  of  two  or  three  days.  The  injections 
may  be  made  intraperitoneally  or  intravenously,  the  latter  being 
preferable.  Not  less  than  nine  days  after  the  injection  the  animal 
is  bled  to  death  from  the  carotid  artery  under  anesthesia,  the  blood 
being  received  in  sterile  test-tubes,  which  are  placed  in  an  inclined 
position  in  the  ice-box.  The  serum  is  removed,  centrifugalized  if  not 
wholly  free  from  blood  corpuscles,  and  placed  in  small  amber  bottles 
with  aseptic  precautions.  These  are  heated  to  56°  C.  for  half  an  hour 
to  effect  inactivation  (to  destroy  complement). 

(6)  Red  Blood  Cells. — Erythrocytes  of  the  sheep  are  used.  The 
blood  of  a  sheep  is  collected  either  in  small  sterile  flasks  containing 
one  volume  of  0.85  per  cent,  salt  solution  and  0.5  per  cent,  sodium 
citrate,  or  in  sterile  centrifuge  tubes.  If  the  former  is  used,  nine 
volumes  of  blood  are  allowed  to  flow  into  the  flask  and  immediately 
mixed  intimately  with  the  citrate  solution,  which  prevents  clotting. 
This  method  is  applicable  if  the  blood  cannot  be  centrifuged  imme- 

1  Fresh  red  blood  cells  of  the  sheep  are  freed  from  serum  by  repeated  washings  with 
physiological  salt  solution — usually  five  washings  "suffice.  The  corpuscles  are  then 
suspended  in  a  volume  of  salt  solution  twice  that  of  the  corpuscles  themselves. 


160    ANTIGENS  AND  THE  TECHNIC  OF  SERUM  REACTIONS 

diately.  If  centrifuge  tubes  are  used,  an  amount  of  blood  not  more 
than  one-third  the  capacity  of  the  tube  (about  5  c.c.)  is  collected  and 
twice  the  volume  of  sterile  salt  solution  is  added  to  it.  The  corpuscles 
are  sedimented,  the  supernatant  solution  is  pipetted  off,  fresh  salt 
solution  is  poured  in,  and  the  corpuscles  resuspended  by  careful 
stirring  with  a  clean  glass  rod.  This  process  is  repeated  five  times, 
each  time  discarding  the  washings.  The  last  time  the  volume  occu- 
pied by  the  erythrocytes  is  read  off  on  the  graduations  of  the  tube 
and  they  are  suspended  in  a  volume  of  salt  solution  twenty  times  that 
occupied  by  the  erythrocytes.  This  makes  a  5  per  cent,  suspension. 
Erythrocytes  are  obtained  by  centrifugalization  from  the  citrated 
blood  in  precisely  the  same  manner.  This  suspension  of  red  blood 
cells,  kept  in  a  cool,  dark  place,  may  be  used  for  two  days,  but  not 
longer.  Beyond  that  time  the  cells  deteriorate  and  hemolyze  with 
abnormal  readiness,  thus  vitiating  the  value  of  the  test. 

(c)  Standardization  of  Hemolytic  System. — It  is  very  important  to 
know  with  exactness  the  amount  of  hemolytic  serum  (inactivated, 
of  course)  which  will  effect  complete  hemolysis  of  1  c.c.  of  a  5  per 
cent,  suspension  of  sheep  erythrocytes  in  the  presence  of  a  constant 
amount  of  complement.  The  determination  of  this  factor  gives  the 
hemolytic  titer  of  the  hemolytic  serum.  It  is  readily  determined 
by  adding  to  a  series  of  tubes,  0.1  c.c.  of  fresh  guinea-pig  serum  (com- 
plement), 1  c.c.  of  erythrocyte  suspension,  and  varying  amounts  of 
the  inactivated  hemolytic  serum.  The  smallest  amount  of  hemolysin 
which  will  effect  hemolysis  under  the  conditions  stated  is  the  hemolytic 
titer  orunit.  Thus,  the  following  tubes  incubated  at  37°  C.  for  one 
hour  showed : 


Result. 
Complete  hemolysis. 


Partial  hemolysis. 
No  hemolysis. 


0.0025  of  this  serum  is  one  unit;  the  hemolytic  titer  is  0.0025  c.c.,  in  other  words. 
It  is  customary  to  use  two  units  in  the  actual  test,  consequently  0.005  c.c.  would  be 
the  amount  used. 


Tube. 

Complement. 

1 

0. 

c.c. 

2 

0. 

c.c. 

3 

0. 

c.c. 

4 

0. 

c.c. 

5 

0. 

c.c. 

6 

0. 

c.c. 

7 

0. 

c.c. 

8 

0. 

c.c. 

9 

0. 

c.c. 

10 

0. 

c.c. 

II1 

0. 

c.c. 

122 

0.0  c.c. 

5  per  cent,  suspension 
sheep  erythrocytes. 

Inactivated 
hemolytic  serum. 

1  C.C. 

0.10         c.c. 

1  c.c. 

0.075      c.c. 

1  c.c. 

0.050      c.c. 

1  c.c. 

0.025      c.c. 

1  c.c. 

0.010      c.c. 

1  c.c. 

0.0075    c.c. 

1  c.c. 

0.0050    c.c. 

1  c.c. 

0.0025    c.c. 

1  c.c. 

0.0010    c.c. 

1  c.c. 

0.00075  c.c. 

1  c.c. 

0.             c.c. 

1  c.c. 

0.             c.c. 

Complement  control. 


2  Erythrocyte  control. 


LYSINS  161 

It  must  be  emphasized  that  precision  of  measurement  is  an  absolute 
requirement  for  success;  the  activating  power  of  complement  for 
hemolysin  does  not  follow  the  law  of  multiple  proportions — it  is  rather 
an  inverse  ratio,  as  Noguchi1  has  pointed  out.  Relatively  less  com- 
plement is  required  to  induce  complete  hemolysis  in  a  system  contain- 
ing four  units  than  is  required  for  a  system  containing  but  a  single 
hemolytic  unit. 

The  serum  to  be  examined  for  specific  antibodies  by  the  method 
of  complement-fixation  must  be  sterile  and  free  from  hemoglobin. 
The  products  of  bacterial  growths  in  serum  may  be  anticomplementary 
and  the  presence  of  hemoglobin  in  serum  also  tends  to  inhibit  hemolysis. 
Blood,  therefore,  should  be  withdrawn  with  aseptic  precautions  from 
the  median  basilic  vein  of  the  patient  into  sterile  test-tubes,  and  either 
centrifugalized  at  once  and  the  serum  removed  from  the  clot,  or 
placed  in  an  inclined  position  in  a  cool  place  until  the  serum  has 
separated.  The  serum  must  be  clear2  and  free  from  erythrocytes  or 
hemoglobin.3  It  is  inactivated  at  54°  to  55°  C.  for  half  an  hour  in  a 
water-bath.4 

The  Technic  of  the  Test. — It  is  essential  that  the  hemolytic  system 
— erythrocytes,  hemolysin,  complement — be  standardized  daily. 
Varying  amounts  of  hemolysin  are  added  to  constant  amounts  of 
erythrocyte  suspension  and  complement,  as  outlined  above.  A 
known  positive  syphilitic  serum  and  a  known  negative  syphilitic 
serum,  together  with  suitable  controls,  must  be  examined  along  with 
the  unknown  serum  to  be  tested. 

The  following  reagents  are  required : 

1.  Sterile  physiological  salt  solution. 

2.  Fresh  guinea-pig  serum  (complement) — add  0.1  c.c.  to  each  tube. 

3.  Five  per  cent,  suspension  of  washed  sheep  erythrocytes  in  normal 
salt  solution — use  1  c.c.  to  each  tube. 

4.  Hemolysin  (amboceptor) — use  twice  the  hemolytic  unit  (the  unit 
must  be  determined  daily). 

5.  Known  syphilitic  serum — inactivate  and  use  0.2  c.c. 

6.  Known  normal  (non-syphilitic)  serum,  inactivated — use  0.2  c.c. 

7.  The  serum  to  be  tested — inactivate,  use  0.2  c.c. 

1  Serum  Diagnosis  of  Syphilis. 

2  Blood  is  best  obtained  early  in  the  morning,  before  the  patient  has  eaten;  blood 
obtained  at  the  height  of  digestion  frequently  contains  fats  which  make  the  serum 
turbid. 

3  Small  amounts  of  blood,  yielding  a  few  drops  of  serum,  may  be  obtained  from  the 
finger-tip  or  the  lobe  of  the  ear.    Massage  must  not  be  practised,  for  there  is  danger  of 
damaging  erythrocytes  with  the  liberation  of  hemoglobin. 

4  Noguchi,  Serum  Diagnosis  of  Syphilis,  states  that  inactivation  at  54°  C.  should  be 
practised — the  higher  temperature  weakens  the  reactive  substance  somewhat. 

11 


162    ANTIGENS  AND   THE   TECHNIC  OF  SERUM  REACTIONS 


Unknown  serum. 

Known  positive 
syphilitic  serum. 

Known  normal 
non-syphilitic  serum. 

Controls. 

Tube  1. 
Serum,  0.2  c.c. 
Complement,  0.1  c.c. 
Salt  solution,  2.7  c.c. 

Tube  3. 
Serum,  0.2  c.c. 
Complement,  0.1  c.c. 
Salt  solution,  2.7  c.c. 

Tube  5. 
Serum,  0.2  c.c. 
Complement,  0.1  c.c. 
Salt  solution,  2.7  c.c. 

Tube  7. 

Complement,  0.1  c.c. 
Salt  solution,  2.9  c.c. 

Tube  2. 
Serum,  0.2  c.c. 
Complement,  0.1  c.c. 
Antigen,1  1  c.c. 
Salt  solution,  1.7  c.c. 

Tube  4. 
Serum,  0.2  c.c. 
Complement,  0.1  c.c. 
Antigen,  1  c.c. 
Salt  solution,  1.7  c.c. 

Tube  6. 
Serum,  0.2  c.c. 
Complement,  0.1  c.c. 
Antigen,  1  c.c. 
Salt  solution,  1.7  c.c. 

Tube  8. 

Complement,  0.1  c.c. 
Antigen,  1  c.c. 
Salt  Solution,  1.9  c.c. 

After  mixing  the  tubes  are  placed  in  a  water-bath  maintained  at 
37°  C.  for  one  hour,  to  permit  of  the  fixation  of  complement;  1  c.c. 
of  a  5  per  cent,  suspension  of  erythrocytes  and  two  units  of  hemolysin 
are  then  added  to  each  tube,  mixed  and  reincubated  for  one  hour, 
then  read.  Tubes  1,  3,  5,  7,  6  and  8  should  show  complete  hemolysis. 
Tube  4  should  show  complete  inhibition  of  hemolysis  (positive  reac- 
tion). If  such  be  the  case  all  the  reagents  are  properly  adjusted,  and 
Tube  2,  containing  the  unknown  serum,  is  read.  If  hemolysis  is 
absent  the  reaction  is  positive;  if  hemolysis  is  complete  the  reaction 
is  negative.2 

The  Method  of  Noguchi.3 — A  rigorous  standardization  of  reagents 
is  a  prerequisite  for  accuracy  in  the  serum  diagnosis  of  syphilis,  and 
Noguchi  has  pointed  out  that  a  variable  inherent  inaccuracy  exists 
in  the  Wassermann  method.  He  has  shown  that  human  sera  may 
contain  variable  amounts  of  hemolysin  specific  for  sheep  erythrocytes. 
Human  sera,  however,  contain  no  hemolysin  for  human  erythrocytes. 
The  Noguchi  modification,  therefore,  substitutes  human  red  blood 
cells  (obtained  from  placenta  or  at  autopsies)  for  sheep  red  blood 
cells.  Rabbits  are  immunized  to  carefully  washed  human  erythro- 
cytes and  the  hemolytic  unit  of  the  rabbit  serum  is  determined  in  the 
usual  manner.  The  following  reagents  are  required  to  perform  the 
Noguchi  test: 

1.  Complement — Fresh  guinea-pig  serum  in  40  per  cent,  dilution 
(one  part  clear  fresh  serum  to  2.5  parts  sterile  salt  solution). 

2.  Hemolytic  Serum — Rabbit   serum,   immunized   against  human 
erythrocytes,  is  titrated  against  human  erythrocytes  to  determine  the 
hemolytic  unit.     Two  units  are  used  in  the  test. 

1  Twice  the  antigen  titer,  determined  by  titration,  diluted  with  salt  solution;  thus,  if 
the  antigenic  titer  of  the  acetone  insoluble  extract  is  0.2  c.c.,  and  the  anticomplementary 
titer  is  found  to  be  1.75  c.c.,  0.4  c.c.  of  the  extract  are  diluted  with  0.6  c.c.  salt  solution 
and  used  in  the  diluted  state.    In  practice,  enough  extract  should  be  diluted  to  last  one 
day. 

2  For  a  discussion  of  results,  see  section  on  Treponema  pallidum. 

3  Noguchi,  Serum  Diagnosis  of  Syphilis. 


PLATE  I 


Wassermann  Reaction.     (Simon.)  * 

A,  positive;  B,  partial;  C,  negative  reaction. 

Note  undissolved  blood  corpuscles  in  A, partial  hemolysis  in  B, and  complete  hemolysis  in  C. 


LYSINS 


163 


3.  Human   Erythrocytes— Red   blood   cells   are   obtained   from   a 
normal  individual,  washed  thoroughly  with  salt  solution,  and  made 
up  as  a  1  per  cent,  suspension  in  salt  solution.    1  c.c.  of  the  suspension 
is  used  in  the  test. 

4.  Antigen — The  acetone-insoluble  antigen  is  used. 

5.  Patient's  Serum — Obtained  fresh,  from  2  to  5  c.c.  of  blood.    It 
is  used  unheated. 

6.  Known  syphilitic  serum. 

7.  Known  normal  (non-syphilitic)  serum. 
The  test  is  performed  as  follows: 


Unknown  serum. 

1 
Known  positive  serum. 

Known  negative  serum. 

Controls. 

Tube  1. 
Serum,  1  drop. 
Complement,1  0.1  c.c. 
Erythrocytes,  1.0  c.c. 

Tube  3. 
j  Serum,  1  drop 
Complement,  0.1  c.c. 
Erythrocytes,  1.0  c.c. 

Tube  5. 
Serum,  1  drop. 
Complement,  0.1  c.c. 
Erythrocytes,  1.0  c.c.' 

Tube  7. 
Complement,  0.1  c.c. 

Erythrocytes,  1.0  c.c. 

Tube  2. 
Serum,  1  drop. 
Complement,  0.1  c.c. 
Antigen,  2  units. 
Erythrocytes,  1.0  c.c. 

Tube  4. 
Serum,  1  drop. 
Complement,  0.1  c.c. 
i  Antigen,  2  units. 
Erythrocytes,  1.0  c.c. 

Tube  6. 
Serum,  1  drop. 
Complement,  0.1  c.c. 
Antigen,  2  units. 
Erythrocytes,  1.0  c.c. 

Mix  and  incubate  one  hour  in  water-bath  at  37°  C.  Remove  and  add  2  units  hemolysin  to  each 
tube  and  incubate  in  water-bath  for  one  hour.  Tubes  1,  3,  5,  6  and  7  should  show  complete  hemo- 
lysis.  Tube  4  should  show  no  hemolysis  (positive  control).  If  such  be  the  case  the  reagents  are 
correctly  adjusted  and  a  reading  of  Tube  2  will  be  positive  (no  hemolysis)  or  negative  (hemolysis). 

A  further  simplification  of  the  method  has  been  made  by  Noguchi. 
The  hemolysin  and  antigen  respectively  may  be  absorbed  on  squares 
of  filter  paper,  dried,  and  standardized.  In  this  state  they  retain 
their  potency  for  several  weeks.  In  practice  the  squares  of  paper  are 
added  directly  to  the  tubes,  thus  saving  much  time. 

Complement-fixation  in  Bacterial  Infections. — Preparation  of  Antigen 
from  Bacteria. — Experience  has  clearly  shown  that  bacterial  antigens 
should  be  polyvalent — prepared  by  mixing  in  equal  amounts,  several 
strains  of  the  same  organism.  The  antigen  may  be  prepared  in  one 
of  several  ways. 

The  simplest  method  is  to  wash  off  bacteria  from  agar  slants,,  at 
the  period  of  maximum  growth,  with  salt  solution  and  shake  thoroughly 
to  make  a  uniform  suspension.  A  small  amount  of  phenol  (0.5  per 
cent.)  and  3  per  cent,  glycerin  are  then  added  and  the  whole  sterilized 
at  56°  to  60°  C.  for  one  hour.  Relatively  more  of  the  proteins  of  the 
bacterial  cell  may  be  obtained  in  solution  if  the  bacterial  emulsion 
is  shaken  in  a  shaking  machine  with  sterile,  sharp  quartz-sand  for 
twenty-four  hours:  filtration  through  coarse  Berkefeld  filters  removes 

1  Forty  per  cent,  solution  of  fresh  guinea-pig  serum  in  salt  solution. 


164  ANTIGENS  AND  THE  TECHNIC  OF  SERUM  REACTIONS 

the  sand  and  broken  bacterial  cells,  and  the  filtrate  is  preserved  with 
0.5  per  cent,  phenol.  Besredka  prepares  a  bacterial  antigen  from  dried 
bacterial  cells,  which  are  obtained  by  drying  bacteria  scraped  from 
agar  slants  or  other  solid  media  over  sulphuric  acid  or  calcium  chloride. 
The  dried  organisms  are  ground  in  agate  mortars  with  crystals  of 
NaCl  to  an  impalpable  powder,  which  is  then  gradually  rubbed  up 
in  successive  portions  of  water  until  a  physiological  salt  solution  is 
obtained  (corresponding  to  8.5  grams  NaCl  in  a  liter  of  distilled  water). 

It  has  been  found  that  much  of  the  antigenic  substance  of  bacteria 
is  precipitated  by  an  excess  of  alcohol;  a  considerable  excess  of  alcohol 
is  added  to  a  suspension  of  bacteria,  or  to  an  emulsion  of  the  cell 
substance  prepared  according  to  Besredka's  process,  outlined  above. 
The  precipitate  from  the  alcoholic  solution  is  separated  by  filtration, 
dried,  and  ground  to  an  impalpable  powder  with  NaCl  crystals. 
The  powder  is  gradually  brought  into  solution  by  the  addition  of 
water  in  successive  amounts  until  isotonicity  is  reached.  An  attempt 
is  made  to  create  a  definite  concentration  of  antigen  by  starting  with 
a  known  quantity  of  dried  bacteria  and  a  corresponding  amount  of 
NaCl  crystals.  Thus,  1  gram  of  dried  bacterial  substance,  ground 
in  a  mortar  with  0.85  gram  NaCl  crystals  and  gradually  brought  to 
a  volume  of  100  c.c.  with  distilled  water,  would  yield,  theoretically, 
an  antigen  of  1  per  cent,  strength.  Bacterial  antigens  must  be  kept 
cold  and  in  a  dark  place,  preferably  in  sealed  amber  bottles.  Deter- 
ioration gradually  occurs  and  all  bacterial  antigens  suspended  or 
dissolved  in  liquids  are  relatively  unstable. 

Standardization  of  Bacterial  Antigens. — The  standardization  of  bac- 
terial antigen  differs  in  no  respect  from  that  of  a  syphilitic  antigen. 
The  anticomplementary  titer  and  the  antigenic  titer  are  determined, 
the  latter  by  titration  with  a  specific  immune  serum. 

The  Diagnosis  of  Glanders  by  the  Method  of  Complement-fixation  — 
The  antigen  is  prepared  from  glycerin-agar  cultures1  of  several  strains 
of  B.  mallei  incubated  at  37°  C.  for  forty-eight  hours.  The  organisms 
are  autolyzed  in  distilled  water  for  several  hours  at  a  relatively  high 
temperature  (70°  to  80°  C.),  then  freed  from  suspended  particles  by 
filtration  through  coarse  Berkefeld  filters.  The  filtrate  is  stored  in 
amber  bottles  in  the  ice-box  after  the  addition  of  0.5  per  cent,  phenol. 

The  anticomplementary  titer  is  determined  from  a  series  of  tubes 
containing  constant  amounts  of  complement  and  graduated  amounts 

1  Reaction  1.5  per  cent,  acid  to  phenolphthalein. 


AGGRESSINS  165 

of  antigen  (1  to  20  dilution  in  salt  solution).1  The  total  volume  of 
complement  and  antigen  is  brought  to  3  c.c.  by  the  addition  of  salt 
solution.  After  one  hour's  incubation  in  the  water  bath  at  37°  C.,  1 
c.c.  of  sheep  erythrocyte  suspension  and  1.5  units  sheep  erythrocyte 
hemolysin  are  added  and  reincubated.  That  dilution  of  antigen  which 
shows  the  slightest  inhibition  of  hemolysis  is  taken  as  the  anti- 
complementary  titer  of  the  antigen.  Not  more  than  one-half  this 
amount,  and  preferably  one-fourth  of  the  anticomplementary  titer, 
is  used  in  the  test. 

The  actual  determination  is  made  in  the  same  manner  as  for  the 
Wassermann  test.2  It  is  well  to  include  a  known  positive  and  known 
negative  glanders  serum  of  the  same  animal  species  as  the  unknown, 
together  with  suitable  controls  of  the  hemolytic  system.  The  length 
of  incubation  is  determined  by  the  time  it  takes  to  effect  complete 
hemolysis  in  the  known  negative  and  the  hemolytic  controls.  Fre- 
quently ten  or  more  hours  will  elapse  before  this  occurs. 

AGGRESSINS. 

Progressively  pathogenic  bacteria  appear  to  differ  from  parasitic 
bacteria  or  "opportunists"  in  that  they  are  able  to  force  an  entrance 
to  the  underlying  tissues  of  the  host  through  natural,  non-specific 
barriers  which  ordinarily  suffice  to  restrain  the  more  parasitic  types 
of  microbes.  Bail3  has  advanced  an  hypothesis,  based  upon  experi- 
mental evidence,  which  attributes  the  invasiveness  of  pathogenic 
bacteria  and  their  ability  to  develop  in  the  tissues  of  the  host  to 
"aggressins."  These  aggressins,  according  to  Bail,  are  present  and 
may  be  demonstrated  in  exudates  resulting  from  bacterial  infection, 
but  they  are  not,  as  a  rule,  found  in  artificial  cultures  of  the  same 
organism.  To  demonstrate  the  action  of  aggressins,  Bail  removed 
bacteria  from  exudates  by  centrifugalization  and  injected  the  clear 
supernatant  fluids,  together  with  a  sublethal  dose  of  the  homologous 
bacterium,  into  experimental  animals.  Rapidly  fatal  infections 
developed.  The  aggressin-containing  exudates  were  not  inactivated 
by  prolonged  exposure  to  50°  C.,  and  it  was  shown,  furthermore,  that 

1  Usually  a  range  of  antigens  from  2  c.c.  to  0.05  c.c.  will  be  found  sufficient. 

2  For  full  discussion  of  results,  see  Mohler  and  Eichhorn,  Bureau  of  Animal  Industry 
Bulletin  136,  April  7,  1911. 

3  See  Der  Problem  der  bakteriellen  Infektion,  Bail,  in  Bibliothek  medizinischer  Mono- 
graphien,  xi;  see  also  Miiller  in  Oppenheimer's  Handbuch  der  Biochemie,  1909,  ii,  1, 
681. 


166    ANTIGENS  AND  THE  TECHNIC  OF  SERUM  REACTIONS 

their  injection  into  susceptible  animals  stimulated  the  formation 
of  "antiaggressin,"  which  greatly  increased  the  resistance  of  the 
animal  to  subsequent  infection.  The  sera  of  animals  immunized 
with  aggressin-containing  fluids  conferred  a  limited  degree  of  immunity 
to  specific  infections  in  non-immune  animals  (passive  immunity). 
It  has  been  claimed  by  Doerr1  and  others  that  the  aggressins  are  of 
the  nature  of  bacterial  endotoxins  and  that  the  immunizing  properties 
of  aggressin  fluids  are  due  to  their  content  of  specific  substances 
derived  from  the  autolysis  of  bacterial  cells. 

The  aggressin  theory  must,  for  the  present,  be  regarded  as  not 
definitely  proved. 

OPSONINS.   TROPINS.   BACTERIAL  VACCINES. 

A  most  important  contribution  to  the  literature  of  immunity  is 
the  work  of  Denys  and  his  associates,2  who  showed  that  the  sera  of 
rabbits  immunized  to  Streptococcus  pyogenes  possessed  two  properties 
not  exhibited  by  the  serum  of  a  normal  animal,  namely,  the  property 
of  restricting  the  development  of  the  organism,  and  the  property  of 
stimulating  phagocytosis.  Their  very  comprehensive  studies  demon- 
strated that  the  leukocytes  of  normal  animals,  suspended  in  the  serum 
of  immunized  animals,  phagocytized  streptococci  energetically,  but 
the  leukocytes  of  immunized  animals  suspended  in  normal  serum 
failed  to  exhibit  phagocytic  activity.  Their  conclusion  was  that  the 
immunity  of  rabbits  to  the  streptococcus  resides  in  the  serum.  These 
observations  not  only  added  materially  to  the  restricted  field  in  which 
they  were  cast — they  brought  sharply  into  focus  the  interrelation  of 
the  humoral  and  cellular  aspects  of  immunity. 

Wright  and  Douglas,3  using  a  modification  of  the  technic  of  Leish- 
man,4  were  able  to  study  phagocytosis  in  vitro:  by  an  ingenious  series 
of  experiments  they  showed  that  normal  serum  contains  substances 
— opsonins — which  prepare  bacteria  for  phagocytosis,  as  described 
in  a  preceding  section  (Cellular  Immunity).  The  technic  of  meas- 
uring the  potency  of  opsonins  in  the  sera  of  normal  and  infected 
individuals,  as  practised  by  Wright  and  his  associates,  consisted 

1  Wien.  klin.  Woch.,  1906,  No.  25. 

2  Denys  and  Le  Clef,  La  Cellule,  1895,  xii;  Bull,  de  1'Acad.  roy.  de  Belgique,  1895; 
Denys  and  Marchand,  Ibid.,  1896;  Van  de  Velde,  Ann.  Inst.  Past.,  1886,  x;  Marchand 
Arch,  de  Med.  exp.,  1898;  Denys,  Cent.  f.  Bakt.,  1898,  xxiv,  685. 

3  See  Studies  in  Immunization,  Constable,  1909,  for  complete  biography. 
«  Brit.  Med.  Jour.,  1902,  i,  73. 


OPSONINS— TROPINS— BACTERIAL   VACCINES  167 

essentially  in  mixing  intimately  equal  volumes  of  bacterial  emulsion, 
serum,  and  leukocytes;  after  incubation  at  body  temperature  the 
mixture  was  spread  evenly  upon  microscopic  slides,  stained,  and 
examined  with  the  microscope.  The  average  number  of  bacteria  per 
polymorphonuclear  leukocyte  was  determined  by  direct  count.  A 
comparison,  under  parallel  conditions,  of  the  phagocytic  activity  of 
leukocytes  for  a  specific  organism  in  the  serum  of  a  normal  individual 
and  that  of  an  individual  infected  with  the  specific  organism,  accord- 
ing to  the  technic  outlined  below,  was  called  by  Wright  the  opsonic 
index. 

Procedure. — 1.  Leukocyte  Suspension. — About  0.5  c.c.  of  blood, 
drawn  from  the  lobe  of  the  ear  or  the  tip  of  the  finger,  is  collected  in 
a  centrifuge  tube  containing  10  c.c.  of  sterile  physiological  salt  solu- 


FIG.  9. — Phagocytosis  of  streptococci. 

tion  in  which  has  been  dissolved  1  per  cent,  of  sodium  citrate;  this 
mixture  is  centrifuged  at  moderate  speed  until  a  sharp  separation 
of  blood  cells  and  clear  supernatant  fluid  is  obtained.  The  super- 
natant fluid  is  carefully  poured  off  and  the  top  layer  of  blood  cells, 
which  contains  practically  all  the  leukocytes,  is  removed  to  a  fresh 
centrifuge  tube  containing  10  c.c.  of  physiological  salt  solution. 

A  second  centrifugalization  is  made,  and  again  the  supernatant 
fluid,  containing  the  last  traces  of  blood  serum,  is  discarded.  The 
sediment,  rich  in  leukocytes,  is  used  as  the  leukocyte  suspension  in 
the  test. 

2.  Suspension  of  Bacteria. — Bacteria  from  a  culture  on  solid  media 
are  suspended  in  sterile  salt  solution  and  agitated  until  a  fine  opales- 
cent emulsion  is  obtained.  This  is  most  conveniently  accomplished 


168     ANTIGENS  AND   THE  TECHNIC  OF  SERUM  REACTIONS 

in  a  shaking  machine,  but  repeated  shaking  in  a  stoppered  test-tube 
containing  glass  beads  will  usually  suffice.  The  coarser  clumps  of 
bacteria  are  removed  by  filtration  through  a  coarse  filter  paper.  The 
density  of  the  bacterial  suspension  should  be  such  that  not  more  than 
ten  bacteria  per  leukocyte  will  be  taken  up  as  the  average. 

3.  Serum. — (a)  Blood  from  three  or  four  normal  individuals  is 
collected  in  capillary  tubes;  after  the  serum  has  separated  a  "pool" 
or  mixture  is  made,  composed  of  equal  volumes  of  each  serum.  Experi- 
ence has  shown  that  "pooled"  serum  furnishes  a  more  reliable  normal 
opsonic  index  than  that  obtained  from  a  single  individual. 

(6)  Serum  from  the  Patient. — This  is  prepared  in  the  manner 
described  above. 

The  Test. — A  capillary  pipette  of  1  to  1.5  mm.  bore  is  made  by 
drawing  out  a  piece  of  glass  tubing  previously  softened  in  the  flame. 
If  the  tubing  is  heated  in  the  center  until  it  softens,  then,  after  a  few 
seconds,  drawn  slowly  and  steadily  out,  the  desired  size  and  shape  is 
readily  obtained.  A  close-fitting  rubber  bulb  attached  to  the  larger 
end  is  a  convenience. 

A  mark  about  1  to  1.5  cm.  from  the  capillary  end  is  made  with  a 
wax  pencil,  and  a  volume  each  of  the  leukocytes,  pooled  serum,  and 
bacterial  suspension  are  drawn  into  the  pipette.  It  is  convenient  to 
separate  each  ingredient  by  a  small  air  bubble,  to  insure  uniformity 
of  volume.  Mixing  is  accomplished  by  carefully  expelling  and  drawing 
back  the  respective  elements  into  the  pipette.  Finally,  the  mixture 
is  drawn  well  up  into  the  pipette,  the  end  is  sealed  in  the  flame  of  a 
Bunsen  burner,  and  the  charged  pipette  is  placed  in  the  incubator 
at  37°  C.  This  is  the  normal  or  control. 

A  precisely  similar  preparation  is  made,  using  the  serum  of  the 
patient  in  place  of  the  pooled  serum. 

Incubation  is  maintained  for  fifteen  minutes. 

The  ends  of  the  pipettes  are  now  broken  off,  and  the  contents  of 
each  pipette  mixed  as  before.  A  large  drop  of  each  respective  mixture 
is  spread  upon  clean  glass  slides,  using  the  same  technic  as  that  for 
preparing  a  blood  smear,  and  air-dried.  The  preparations  are  stained 
with  Loffler's  methylene  blue,  Wright's  stain,  or  other  stain  suitable 
for  the  organism  used. 

The  number  of  bacteria  in  fifty,  one  hundred,  or  two  hundred  leuko- 
cytes are  determined  by  direct  count,  and  the  average  number  of 
bacteria  per  leukocyte  of  the  normal  serum  compared  with  the  average 
number  of  bacteria  per  leukocyte  in  the  pathological  serum: 


OPSONINS— TROPINS— BACTERIAL   VACCINES  169 

EXAMPLE. 

Bacteria  in  Bacteria  per 

100  leukocytes.  leukocyte. 

Staphylococcus   suspension    +    pooled   serum   and 

leukocytes 750  7.5 

Staphylococcus  suspension   +  patient's  serum  and 

leukocytes 250  2.5 

2  5 

Opsonic  index,  patient's  serum  =  — '--  or  0.33  per  cent. 

7 . 5 

Numerous  observers  have  been  unable  to  obtain  uniform  results 
with  the  technic  of  Wright  for  opsonic  index  determination,  and  this 
is  not  surprising  when  the  many  variable  factors  entering  into  the 
method  are  reviewed.  Attempts  have  been  made  to  eliminate  or 
limit  the  variable  factors:  Simon  proposed  a  dilution  method  in 
which  the  pooled  and  patient's  serum  are  diluted  1  to  10,  1  to  100, 
etc.,  before  incubation  with  the  bacteria  and  leukocytes.  That  dilu- 
tion of  serum  at  which  phagocytosis  practically  ceases  in  the  normal 
and  patient's  serum  respectively  is  taken  as  a  basis  for  comparison. 
Inasmuch  as  the  opsonic  index  is  rarely  determined  as  a  guide  for 
treatment  of  bacterial  disease  with  bacterial  vaccines  at  the  present 
time,  however,  a  discussion  of  these  modifications,  which  are  too 
involved  for  practical  use,  is  left  for  more  pretentious  volumes. 

The  Nature  of  Opsonins. — There  appears  no  doubt  that  the  hypo- 
thetical substance  or  substances  called  opsonin  by  Wright  exist  in 
normal  sera,  and  it  is  equally  certain  that  they  may  be  diminished 
during  infection.  Furthermore,  opsonin  may  be  increased  either  in 
amount  or  in  potency  by  careful  immunization.  The  relation  of 
opsonins  to  other  antibodies,  normal  or  specific,  is  a  subject  of  con- 
troversy at  present.  The  researches  of  Neufeld  and  Rimpau,1  Hek- 
toen2  and  others  indicate  that  the  normal  opsonins — those  of  normal 
sera — are  thermolabile,  but  those  developed  during  immunization 
to  a  specific  organism — bacteriotropins — are  relatively  thermostabfte. 

It  has  been  suggested  that  opsonins  or  bacteriotropins  are  not  to 
be  distinguished  from  other  immune  bodies — as  normal  and  specific 
amboceptors  or  agglutinins.  The  rapidity  with  which  the  opsonic 
index  may  be  increased  or  diminished  within  a  few  hours  following 
injections  of  bacteria,  however,  would  suggest  a  possible  distinction 
between  these  antibodies  and  the  slowly  developing  specific  bacteri- 
cidal and  agglutinating  antibodies. 

Vaccine  Therapy. — The  value  of  vaccines  and  of  autogenous  vac- 
cination in  bacterial  prophylaxis  and  bacterial  immunization  as  set 

1  Deutsch.  med.  Wchnschr.,  1904,  1458. 

2  Jour.  Inf.  Dis.,  1906,  iii,  434;  1909,  vi,  78;  1913,  xii,  1. 


170     ANTIGENS  AND  THE   TECHNIC  OF  SERUM  REACTIONS 

forth  by  Wright  marks  a  distinct  epoch  in  bacterial  therapeutics  in 
spite  of  the  practical  failure  of  his  opsonic  index  determination  as 
a  theoretical  guide  to  immunization  and  treatment.  He  has  used 
bacterial  vaccines  both  for  prophylaxis — to  prevent  infection  with 
specific  bacteria — and  therapeuitically— to  arrest  infection. 

Prophylactic  Vaccination. — The  object  of  prophylactic  vaccination  is 
to  increase  the  resistance  of  the  recipient  to  specific  infection.  This 
is  accomplished  by  reinforcing  the  natural  initial  defenses  of  the 
body  with  specific  antibodies,  generated  in  the  host  in  response  to 
the  injection  of  the  specific  microorganism  as  a  vaccine.  In  prophy- 
lactic vaccination  the  host  has  ample  time  to  work  over  the  vaccine, 
and  by  prolonging  the  treatment  through  repeated  graduated  doses 
the  maximum  degree  of  immunity  may  be  expected.  To  attain  the 
maximum  immunizing  effect  the  bacteria  of  the  vaccine  should  be  as 
near  their  normal  state  as  possible,  that  is,  they  should  be  endowed 
with  all  the  antigenic  properties  they  possess  in  the  natural  disease 
produced  by  them  in  the  host. 

Following  the  brilliant  work  of  Jenner  with  cowpox  vaccine  and  the 
epoch-making  observations  of  Pasteur,  observers  are  fairly  agreed- 
that  the  best  results  from  prophylactic  vaccination  are  obtainable 
only  by  the  use  of  an  attenuated  living  virus.  The  action  of  such  a 
living  virus  is,  as  Theobald  Smith1  has  aptly  expressed  it,  "  a  multitude 
of  feeble  blows,  each  of  which  produces  an  immunological  response." 
The  dangers  attending  the  use  of  attenuated  viruses,  however,  ordi- 
narily preclude  their  employment,  due  to  inability  to  control  the 
virulence  of  attenuated  cultures.  The  possibility  of  creating  carriers 
cannot  be  overlooked.  For  this  reason  killed  cultures  are  almost 
invariably  selected. 

It  is,  of  course,  impossible  to  utilize  an  autogenous  vaccine,  but 
for  purposes  of  immunization  a  polyvalent  vaccine  is  indicated.  The 
action  of  a  dead  virus  is  limited  practically  to  a  single  immunological 
response,  hence  the  need  of  repeated  inoculations. 

Therapeutic  Vaccination. — In  chronic,  long-drawn  out  focal  or  local 
infections,  the  invading  microbes  are  either  holding  their  own  or 
gaining  the  ascendency  and  the  object  of  bacterial  vaccination  is  to 
turn  the  tables  on  the  invaders.  The  products  of  immunization  must 
be  used  at  once,  arid  the  organisms  comprising  the  vaccines  for  this 
purpose  cannot  ordinarily  be  as  resistant  as  their  originals  in  the  host. 
The  underlying  principle  of  therapeutic  vaccination,  according  to 

i  Jour.  Am.  Med.  Assn.,  1913,  Ix,  1591. 


OPSONINS— TROPINS— BACTERIAL  VACCINES  171 

Wright,1  is  to  exploit  the  normal  tissues  of  the  body  in  the  interest 
of  the  infected  tissue.  For  this  purpose,  microbes  similar  to  those 
causing  the  infection  (autogenous  organisms)  are  inoculated  into 
some  other  part  of  the  body.  This  inoculation  is  not,  to  use  Wright's 
phraseology,  a  mere  replica  of  the  original  infection;  there  are  two 
important  points  of  difference:  (1)  the  microbes  of  the  vaccine  are 
killed,  so  that  their  multiplication  within  the  host  is  impossible;  (2) 
the  dose  of  vaccine  must  be  so  regulated  that  the  tissues  of  the  host 
at  the  site  of  inoculation  and  elsewhere  must  inevitably  win.  Victory 
of  the  host  is  brought  about  through  the  elaboration  of  specific  anti- 
bodies generated  in  the  healthy  tissues  on  a  scale  more  than  adequate 
to  bring  about  a  destruction  of  the  organisms  introduced  into  the 
healthy  tissue.  The  surplus  of  the  specific  antibodies  will  find  its 
way,  through  blood  and  lymph  channels,  to  the  focus  of  infection, 
and  will  reinforce  the  partially  depleted  defensive  forces  which  have 
ineffectually  opposed  the  invading  organisms. 

It  should  be  borne  in  mind  that  vaccine  therapy  cannot  be  reason- 
ably applied  unless  an  exact  bacteriological  diagnosis  has  been  made. 
The  immunizing  effects  of  vaccines  are  definitely  limited  by  the 
ability  of  the  normal  tissues  of  the  patient  to  produce  antibodies; 
to  inject  too  frequently  or  in  too  large  doses  may  not  only  be  barren 
of  results — it  may  result  in  a  decrease  rather  than  an  increase  of 
resistance  to  infection. 

It  is  essential  for  the  best  results  of  vaccination  that  the  focus  of 
infection  be  so  situated  anatomically  that  the  newly  formed  antibodies 
be  drawn  to  the  infected  area  by  the  production  of  local  hyperemia. 
Infections  of  long  standing  naturally  respond  to  treatment  more 
slowly  than  newly  acquired  infections. 

Preparation  of  Vaccines. — Much  discussion  has  arisen  concerning 
the  use  of  autogenous  vaccines  as  compared  with  stock  or  polyvalent 
vaccines.  So  little  is  actually  known  of  what  vaccines  may  accomplish 
in  the  body  that  it  is  impossible  to  answer  this  question  definitely. 
It  is  desirable,  however,  to  retain  in  the  vaccine  all  possible  anti- 
genie  properties  which  were  possessed  by  the  organism  in  the  body. 
It  is  a  well-known  fact  that  certain  kinds  of  organisms  rapidly  lose 
their  ability  to  produce  disease  when  they  are  grown  for  any  length 
of  time  outside  the  body.  Others  retain  their  virulence  for  some 
time.  This  would  appear  to  indicate  that  stock  vaccines  of  the  former 
would  be  unsatisfactory,  while  stock  vaccines  of  the  latter  might  be 

1  Proc.  Roy.  Soc.  of  Med.,  London,  1910,  iii. 


172  ANTIGENS  AND  THE  TECHNIC  OF  SERUM  REACTIONS 

more  successful.    It  is  a  safe  general  rule  to  state  that  an  autogenous 
vaccine  is  desirable. 

The  preparation  of  vaccine  is  carried  out  as  follows: 

1.  Obtain  pure  cultures  of  the  organisms  from  the  lesion  or  what- 
ever material  is  available.    The  details  of  culture  vary  with  the  type 
of  organism  that  is  expected. 

2.  Inoculation  of  the  pure  culture,  or  cultures  in  the  event  of  mul- 
tiple infection,  in  suitable  media  to  furnish  the  desired  amount  of 
growth. 

3.  Removal  of  the  growth,  with  sterile  precautions,  to  a  sterile 
container,  such  as  a  test-tube  containing  sterile  glass  beads.     This 
is  accomplished  by  washing  the  growth  from  the  medium  into  sterile 
saline  solution :  5  to  1 0  c.c.  of  salt  solution  are  required  for  an  ordinary 
agar  slant  culture.    When  enough  growth  is  accumulated  it  is  trans- 
ferred to  the  sterile  test-tube,  being  careful  that  no  organisms  con- 
taminate the  upper  part,  else  they  may  escape  sterilization. 

4.  Sterilization:     Heat  the  bacterial  suspension  in  a  water-bath. 
Usually  one  hour  at  60°  to  65°  C.  suffices.    Care  must  be  taken  that 
the  level  of  water  in  the  water-bath  is  well  above  that  of  the  level 
of  the  suspension  in  the  test-tube. 

5.  Test  sterility  of  the  suspension.    Inoculate  suitable  media  and 
observe  the  absence  of  growth.     In  skin  infections  it  is  sometimes 
desirable  to  exclude  the  presence  of  the  tetanus  bacillus. 

6.  Shake  the  suspension  vigorously  to  distribute  the  organisms 
uniformly  in  it. 

7.  Standardize:     Determine  the  number  of  bacteria  in  a  cubic 
centimeter.    This  is  very  simply  accomplished  by  thoroughly  mixing 
equal  volumes  of  freshly  drawn  blood  and  bacterial  emulsion  in  a 
pipette,  spreading  the  mixture  on  a  microscope  slide,  drying  and 
staining  it  with  Wright's  or  Jenner's  stain.     Determine  by  actual 
counting  in  a  number  of  fields  the  proportion  of  bacteria  to  red  cells. 
Knowing  the  number  of  red  blood  cells  in  a  cubic  centimeter  of  blood 
(5,000,000,000)  and  the  proportion  of  bacteria  to  red  blood  cells,  it  is  a 
simple  matter  to  determine  the  number  of  bacteria  in  the  suspension. 

A  more  accurate  procedure  is  to  draw  up  one  volume  of  vaccine  in 
the  erythrocyte  pipette  of  a  hemocytometer,  dilute  to  the  101  mark 
with  a  dilute  solution  of  fuchsin  or  other  suitable  stain,  mix  and 
transfer  to  the  counting  chamber.  An  enumeration  of  the  bacteria 
is  made  in  precisely  the  same  manner  that  a  blood  count  is  made. 


OPSONINS— TROPINS— BACTERIAL   VACCINES  173 

8.  Dilute  the  suspension  to  the  required  degree  with  phenol,  so 
that  the  finished  vaccine  shall  contain  0.25  to  0.5  per  cent,  of  it.    This 
is  the  finished  vaccine. 

9.  Redetermine  sterility  if  necessary. 

Sensitized  Vaccines. — Killed  bacteria  which  have  been  immersed 
in  a  specific  serum — sensitized  vaccines — are  said  to  be  less  liable 
to  produce  general  and  local  reactions.  The  immunity  developed 
in  response  to  the  injection  of  these  sensitized  vaccines  is  said  to 
appear  more  rapidly,  and  doses  thirtyfold  those  of  unsensitized  vac- 
cines may  be  injected  without  serious  effect. 

The  Injection. — The  skin  at  the  site  of  injection  is  cleaned  with 
soap  and  water  and  then  with  alcohol;  or  better,  after  carefully  dry- 
ing it  is  painted  with  tincture  of  iodin.  The  required  amount  of 
vaccine  is  injected  subcutaneously  through  this  area,  from  a  sterile 
syringe. 

The  Dosage  and  Frequency  of  Injection. — It  is  advisable  to  begin 
with  small  doses  of  vaccine,  quantities  which  past  experience  has 
shown  to  do  no  harm  so  far  as  can  be  determined  by  clinical  evidence, 
and  to  increase  the  size  of  the  dose  gradually,  the  injections  usually 
being  given  at  intervals  of  about  a  week.  If  no  change  results  from 
the  treatment,  larger  doses  may  be  tried.  If  the  symptoms  become 
aggravated  the  doses  should  be  diminished  and  given  at  less  frequent 
intervals.  Generally  speaking,  in  the  more  acute  cases  smaller  doses 
should  be  selected  to  begin  with,  larger  doses  being  reserved  for  the 
more  chronic  cases.  The  amounts  of  vaccine  to  be  injected  vary  widely 
according  to  different  investigators.  Generally  speaking,  the  following 
figures  are  fairly  representative : 

Minimum.1  Maximum.1  Average.1 

Staphylococcus    ......  5.0  1000  25 

Streptococcus 2.5  100  25 

Pneumococcus 2.5  100  25 

Goriococcus 2.5  300  30 

Coli 5.0  1000  100 

Pyocyaneus    ........  5.0  1000  100 

Indications  for  the  Use  of  Bacterial  Vaccine. — Generally  speaking, 
bacterial  vaccines  are  contraindicated  in  acute  disease,  but  may  be 
employed  in  practically  any  localized  infection,  or  an  infection  which 
has  become  chronic.2 

1  Figures  represent  millions  of  organisms. 

2  An  excellent  discussion  of  the  present  status  of  vaccine  therapy  is  that  of  Theobald 
Smith,  An  Attempt  to  Interpret  the  Present-day  Use  of  Vaccines,  Jour.  Am.  Med.  Assn., 
1913,  Ix,  1591. 


174  ANTIGENS  AND  THE  TECHNIC  OF  SERUM  REACTIONS 

Results. — Opinions  differ  widely  as  to  the  value  of  vaccines.  Accord- 
ing to  the  theory  of  bacterial  vaccination,  subacute  and  chronic  infec- 
tions which  are  localized  should  give  the  best  results,  and  such  indeed 
appears  to  be  the  case.  For  example,  a  streptococcus  septicemia 
abates  and  leaves  a  joint  involvement  or  a  heart  valve  vegetation. 
Vaccine  therapy  has  a  better  chance  of  producing  results  during  this 
secondary  stage  than  during  the  earlier  acute  septicemic  stage.  Gon- 
orrheal  arthritis,  pneumonias  which  resolve  by  lysis,  pus  sinuses,  and 
localized  colon  infections  are  suitable  for  treatment.  In  acute  inflam- 
mations of  the  mucous  membranes  of  the  intestines,  bladder,  throat, 
etc.,  the  results  have  been  either  negative  or  unsatisfactory. 

So  far  as  specific  organisms  are  concerned,  staphylococcus  vaccines 
give  the  most  constant  and  satisfactory  results.  Furuncles,  severe 
carbuncles,  some  cases  of  acne,  and  even  low-grade  staphylococcus 
septicemias  yield  rather  readily  to  vaccine  therapy  with  this  organism. 
Streptococcic  and  pneumococcic  infections  are  much  more  resistant, 
generally  speaking,  to  vaccine  treatment  than  are  staphylococcus 
infections. 


CHAPTER   IX. 

THE  MICROSCOPIC  AND  CULTURAL  STUDY  OF 
BACTERIA. 


2.  Capsules. 

3.  Polar  Bodies. 

4.  Flagella. 

F.   Differential  Stains  for  Bac- 
teria. 

1.  Gram. 

2.  Ziehl-Neelsen. 

3.  Gabbett. 

4.  Polychrome.  Stains. 

B.  Preparation  of  Stains.  5.  Smith  Sputum  Stain. 

C.  Technio  of    Staining   Bac-       III.  STAINING  BACTERIA  IN  TISSUES. 


METHODS  FOR  THE  MICROSCOPIC  STUDY 

OF  BACTERIA. 
I.  LIVING  BACTERIA. 

A.  Hanging  Drop. 

B.  Hanging  Block. 

C.  Dark  Ground  Illumination. 

D.  Intra  Vitam  Staining. 
II.  STAINING  OF  BACTERIA. 

A.   Chemistry  of  Stains. 


teria. 

D.  Intensive   Stains   for   Bac- 
teria. 


IV.  METHODS  AND  MEDIA  FOR  THE 

CULTIVATION  OF  BACTERIA. 
V.  CULTIVATION  OF  BACTERIA. 


E.   Stains    for    Special    Struc- 1  Inoculation  of  Cultures. 


tures    of    the    Bac- 
terial Cell. 


1.  Spores. 


Isolation  of  Pure  Cultures. 
Incubation   of   Cultures. 
VI.  STUDY  OF  BACTERIAL  CULTURES. 


METHODS   FOR   THE   MICROSCOPIC   STUDY   OF   BACTERIA. 

BACTERIA  may  be  examined  directly  under  the  higher  powers  of  the 
microscope  for  their  morphology,  motility,  arrangement,  method  of 
reproduction,  and  their  behavior  in  specific  sera,  or  they  may  be 
stained  with  various  anilin  dyes  and  chemicals  to  bring  out  details 
of  structure  or  composition,  and  their  relation  to  various  tissues  in 
pathological  processes. 

Glass  slides  and  cover-glasses  are  conveniently  used  for  this  purpose. 
Microscopic  slides  should  be  made  from  clear,  colorless  glass.  Cover- 
glasses  should  be  made  of  thin  glass.  The  available  working  distance 
of  oil-immersion  lenses  is  somewhat  less  than  1.5  mm.,  consequently 
cover-glasses  should  not  measure  more  than  1  mm.  in  thickness  as 
a  maximum  limit.  Number  1  cover-glasses  are  suitable  for  bacterio- 
logical work. 

Glass  slides  and  cover-glasses  are  best  cleaned  in  a  mixture  of 
potassium  bichromate,  1  part;  water,  4  parts;  sulphuric  acid,  6  parts. 
The  bichromate  is  dissolved  in  the  water  with  the  aid  of  heat  and 
cooled;  the  acid  is  added  slowly  with  constant  stirring.  Immersion 
in  this  mixture  for  twenty-four  hours  removes  dirt  and  grease  from 


176     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

both  slides  and  cover-glasses.  The  cleaned  glassware  is  removed  from 
the  cleansing  bath  and  washed  with  running  water  until  neutral  to 
litmus  paper.  It  is  stored  either  in  slightly  ammoniacal  alcohol,  or 
dried  with  a  soft  cloth,  previously  freed  from  grease  by  boiling  in  a 
5  per  cent,  sodium  carbonate  solution. 

I.  Examination  of  Living  Bacteria. — A.  Hanging  Drop. — The  motil- 
ity,  shape,  and  size  of  bacteria  may  be  studied  in  a  "hanging-drop" 
preparation.  A  drop  of  fluid  from  a  bacterial  culture  in  liquid  media 


FIG.  10. — Hollow-ground  slide  for  hanging  drop. 

is  transferred  to  the  center  of  a  thin  cover-glass.  If  the  growth  is 
upon  solid  media  a  drop  of  physiological  salt  solution1  is  placed  upon 
the  center  of  the  cover-glass  as  before,  and  a  very  small  amount  of 
the  culture  is  removed  with  a  platinum  needle  and  emulsified  in  it. 
Next,  the  rim  of  the  concavity  in  a  "  hollow-ground  slide"  is  ringed 
with  vaselin  and  the  cover-glass  is  inverted  over  it  in  such  a  manner 
that  the  drop  is  suspended  in  the  hollow,  but  touches  neither  the 
sides  not  the  bottom.  The  vaselin  seals  the  preparation,  causing  it 
to  adhere  to  the  slide,  and  also  prevents  evaporation.  The  prepara- 
tion is  now  ready  for  microscopic  examination.  The  one-sixth  or  one- 
eighth-inch  objective  should  be  used,  with  the  diaphragm  partly  closed 
to  reduce  the  intensity  of  illumination.  It  is  desirable  to  focus  first 
upon  the  edge  of  the  drop;  the  edge  is  sharply  defined  and  readily 
located.  Bacteria  are  usually  more  numerous  at  the  edge  than  in  the 
center  of  the  drop. 

B.  Hanging  Block. — It  is  desirable  occasionally  to  follow  the 
development  of  bacteria  through  several  generations,  to  study  the 
germination  of  spores,  or  to  examine  special  structures  within  the 
bodies  of  individual  organisms.  The  hanging-drop  method  is 
unsuited  for  this  purpose,  which  presupposes  immobilization  of  the 
organism.  Hill2  has  invented  an  ingenious  modification  of  the  hang- 
ing-drop method,  the  hanging  block,  which  fulfils  this  requirement. 
His  directions  for  preparing  it  are: 

"Pour  melted  nutrient  agar  into  a  Petri  dish  to  the  depth  of  about 
one-eighth  or  one-quarter  inch.  Cool  this  agar  and  cut  from  it  a  block 

1  Physiological  salt  solution  is  prepared  by  dissolving  8.5  grams  NaCl  in  distilled 
water  1000  c.c. 

2  Jour.  Med.  Research,  March,  1902,  vii,  202. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      177 

about  one-quarter  inch  to  one-third  inch  square  and  of  the  thickness 
of  the  agar  layer  in  the  dish.  This  block  has  a  smooth  upper  and 
under  surface.  Place  it,  under  side  down,  on  a  slide  and  protect  it 
from  dust.  Prepare  an  emulsion,  in  sterile  water,  of  the  organism  to 
be  examined  if  it  has  been  grown  on  a  solid  medium,  or  use  a  broth 
culture;  spread  the  emulsion  or  broth  upon  the  upper  surface  of  the 
block  as  if  making  an  ordinary  cover-slip  preparation.  Place  the 
slide  and  block  in  a  37°  C.  incubator  for  five  to  ten  minutes  to  dry 
slightly.  Then  lay  a  clean  sterile  cover-slip  on  the  inoculated  surface 
of  the  block -in  close  contact  with  it,  carefully  avoiding  air-bubbles. 
Remove  the  slide  from  the  lower  surface  of  the  block  and  invert 


FIG.  11. — Warm  stage,  electrically  heated,  for  the  cultivation  of  bacteria. 

the  cover-slip  so  that  the  agar  block  is  uppermost.  With  a  platinum 
loop,  run  a  drop  or  two  of  melted  agar  along  each  side  of  the  agar 
block,  to  fill  the  angles  between  the  sides  of  the  block  and  the  cover- 
slip.  This  seal  hardens  at  once,  preventing  slipping  of  the  block. 
Place  the  preparation  in  the  incubator  again  for  five  or  ten  minutes, 
to  dry  the  agar-agar  seal.  Invert  this  preparation  over  a  moist  4. 
chamber  and  seal  the  cover-slip  in  place  with  white  wax  or  paraffin. 
Vaselin  softens  too  readily  at  37°  C.,  allowing  shifting  of  the  cover- 
slip.  The  preparation  may  then  be  examined  at  leisure.1 

1  A  light,  detachable,  electrically  heated  warm-stage  incubator,  manufactured  by 
the  Chicago  Surgical  and  Electrical  Company  according  to  specifications  furnished 
by  the  writer  is  very  satisfactory  for  this  purpose.  Bacteria  may  be  maintained  con- 
stantly at  any  desired  temperature  between  that  of  the  room  and  45°  C.  for  several 
days,  and  observed  continuously  without  difficulty.  If  the  warm-stage  incubator  is 
attached  to  a  graduated  mechanical  stage,  many  individual  bacteria  may  be  observed 
in  the  same  preparation  by  recording  their  respective  positions  as  indicated  on  the 
graduated  rectilinear  stage  verniers. 
12 


178     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

C.  Dark  Field  Illumination  and  Ultramicroscopic  Examination. — For 
the  study  of  very  minute  particles  in  suspension,  the  ultramicroscope 
of  Siedentoff  and  Zsigmondy1  has  been  used,  but  the  dark-ground 
illumination  apparatus  of  Reichert,2  a  much  simpler  device,  readily 
adjusted  to  any  microscope,  has  largely  supplanted  it  for  bacterial 
examinations.     With  the  Reichert  apparatus  the  flagella  of  bacteria 
and  other  structures  of  low-refractive  index  may  be  observed.    Tre- 
ponema  pallidum  in  fresh  smears  from  lesions  is  readily  seen  with  the 
dark  ground  illuminating  apparatus. 

D.  Intra  Vitam  Staining. — Nakanishi3  has  applied  the  method  of 
infra  vifam  staining  to  the  study  of  spores  and  granules  in  living  bac- 
terial cells.     The  method  consists  essentially  in  emulsifying  a  small 
amount  of  bacterial  growth  in  normal  salt  solution  containing  suffi- 
cient aqueous  methylene  blue  to  impart  a  distinct  blue  color  to  the 
solution.     The  preparation  is  viewed  as  in  the  hanging-drop  slide. 
The  organisms  absorb  sufficient  dye  to  impart  to  them  a  faint  color, 
and  granules  within  their  bodies  frequently  stain  with  moderate  inten- 
sity.   The  development  of  spores  from  pre-sporogenic  granules  may  be 
studied  by  this  method. 

II.  Staining  of  Bacteria. — A.  Chemistry  of  Stains. — The  stains  of 
value  for  coloring  bacteria  are  almost  exclusively  anilin  dyes  which 
contain  one  or,  more  commonly,  several  benzene  rings.  Their  color- 
ing properties  have  been  shown  to  depend  upon  two  distinct  radicals; 
double-bonded  atoms  as  C  =  C,  C  =  O,  C  =  N,  N  =  N,  known  as 
chromophoric  groups,  and  auxochromic  groups,  which  impart  to  or 
intensify  the  color.  Of  the  chromophoric  groups,  NH2  and  —OH  are 
the  more  important.  The  latter  form  salts  which  may  be  either  basic 
or  acid  in  character.  Bacteria  usually  stain  best  with  basic  dyes,  as 
do  nuclei  of  higher  plant  and  animal  cells. 

The  chemistry  of  the  staining  process  itself  is  a  matter  of  discussion. 
It  was  formerly  held  that  the  cell  protoplasm  united  chemically  with 
the  stain  as  an  acid  unites  with  a  basic  salt,  but  later  investigations, 
particularly  those  of  Michaelis,4  are  not  in  harmony  with  this  view. 
It  is  probable  that  the  physical  state  of  the  cell  membrane  as  well 
as  the  composition  of  the  cytoplasm  play  a  part  in  the  staining  process. 

B.  Preparation  of  Stains. — Stains  prepared  by  Griibler  or  Merck 
are  commonly  used  for  the  staining  of  bacteria.  They  are  conveniently 

1  Zeit.  f.  wissenschaftl.  Mikroskopie,  1909,  xxvi,  391. 

2  Miinchen.  med.  Wchnschr.,  1906,  2351;  Hyg.  Rund.,  1907,  No.  18;  Cent.  f.  Bakt. 
Orig.,  1909,  li,  14. 

3  Miinchen.  med.  Wchnschr.,  1900,  No.  6;  Cent.  f.  Bakt.,  1901,  xxx,  97,  145,  193,  225. 

4  Einfiihrung  in  die  Farbstoffchemie,  1902,  Berlin. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      179 

kept  in  stock  as  saturated  aqueous  or  alcoholic  (96  per  cent.)  solutions. 
The  solubility  of  stains  in  water  and  in  alcohol  respectively  varies, 
but,  as  a  rule,  the  solubility  in  alcohol  is  greater  than  that  in  water. 
Saturated  solutions  of  anilin  dyes  are  unsuited  for  the  staining  of 
microorganisms,  but  they  are  more  stable  than  diluted  solutions 
provided  they  are  kept  in  tightly-stoppered  bottles  away  from  the 
light.  Dilutions  of  saturated  solutions  are  prepared  as  they  are 
needed  for  current  use. 

C.  Technic  of  Staining  Bacteria. — 1.  Preparation  of  a  film  of  bac- 
teria for  staining:    A  drop  of  a  culture  of  bacteria  from  a  fluid  medium 
— as  broth — is  removed  with  a  platinum  loop  and  spread  upon  a  clean 
cover-glass  or  glass  slide.     Bacteria  from  a  solid  medium  are  emul- 
sified in  a  small  drop  of  water  on  the  slide.1 

2.  The  film  of  bacteria  is  allowed  to  dry  in  the  air;  evaporation 
may  be  hastened  in  the  incubator  at  37°  C. 

3.  The  air-dried  film  is  next  fixed  by  passing  it  once  slowly  through 
the  flame  of  a  Bunsen  burner,  film  side  upward;  about  one-half  second's 
exposure  to  the  flame  suffices;  a  longer  exposure  destroys  or  changes 
the  staining  properties  of  the  organisms. 

4.  Staining:     A  5  per  cent,  aqueous  solution  of  methylene  blue, 
fuchsin,  or  gentian  violet,  prepared  by  adding  5  c.c.  of  filtered  satu- 
rated stock  solution  to  95  c.c.  of  distilled  water,  is  used.    The  slide 
or  cover-glass  is  flooded  with  the  desired  stain,  and  after  one  to  five 
minutes,  depending  upon  the  intensity  of  the  stain  used,  the  excess 
is  poured  off  and  the  preparation  is  washed  thoroughly  with  water. 
The  residual  moisture  is  removed  with  filter  paper  or  by  air-drying, 
and  a  small  drop  of  Canada  balsam  (dissolved  in  xylol)  is  placed  in 
the  center  of  the  stained  area.     The  film  is  finally  enclosed  between  a 
slide  and  a  cover-glass. 

D.  Intensive  Stains  for  Bacteria. — Simple  aqueous  or  alcoholic  solu- 
tions of  anilin  dyes  are  frequently  inefficient  for  staining  bacteria  and 
resort  is  made  to  intensified  stains.     One  of  the  most  useful  of  the 
intensified  stains  is  Loffler's  alkaline  methylene  blue,  prepared  in  the 
following  manner: 

1  to  10,000  aqueous  solution  of  potassium  hydroxide2 70  c.c. 

Saturated  alcoholic  solution  methylene  blue 30  c.c. 

1  It  is  essential  that  the  emulsion  shall  be  but  faintly  opalescent  when  viewed  by 
reflected  light;  a  distinct  clouding  indicates  that  too  many  organisms  have  been  added, 
in  which  event  the  preparation  will  be  found  to  be  unsatisfactory. 

2  Conveniently  prepared  by  dissolving  1  gram  of  KOH  in  100  c.c.  distilled  water 
and  adding  1  c.c.  of  this  solution  to  99  c.c.  of  distilled  water. 


180     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

Fixed  films  of  bacteria  are  stained  from  one  to  five  minutes  with 
this  stain,  or  the  films  are  flooded  with  the  stain  and  heated  until 
steam  rises  (not  bbiled)  for  one  to  three  minutes.  It  is  difficult  to 
overstain  with  Loffler's  methylene  blue  unless  evaporation  takes 
place  to  such  a  degree  that  the  stain  dries  on  the  slide.  The  stain  is 
washed  off  with  water,  dried,  and  mounted. 

E.  Stains  for  Special  Structures  of  the  Bacterial  Cell. — 1.  Spores. — 
(a)  Flood  fixed  film  of  bacteria  with  carbol-fuchsin1  and  steam  (not 
boil)  for  five  minutes. 

(6)  Wash  thoroughly  in  running  water. 

(c)  Decolorize  with  1  per  cent,  sulphuric  acid  until  excess  stain  is 
removed. 

(d)  Wash  thoroughly  in  running  water. 

(e)  Flood   with   saturated   aqueous   solution   methylene   blue    (or 
Loffler's  alkaline  methylene  blue)  and  allow  to  stain  one  minute. 

(/)  Wash  in  water,  dry,  and  mount. 
Spores  stain  red,  vegetative  cells  blue. v 

Holler's  Spore  Stain:2 — (a)  Suspend  the  fixed  film  of  bacteria  in 
chloroform  for  two  minutes. 
(6)  Wash  with  water. 

(c)  Flood  with  5  per  cent,  chromic  acid  solution  for  two  minutes. 

(d )  Wash  thoroughly  in  running  water. 

(e)  Flood  with  carbol-fuchsin  and  steam  for  five  minutes. 
(/)  Wash  thoroughly  in  water.  . \*  i 

(g)  Decolorize  with  1  per  cent,  sulphuric  acid  until  excess  stain  is 
removed. 

(h)  Wash  thoroughly  in  water. 

(i)  Flood  with  Loffler's  alkaline  methylene  blue  and  allow  to  stain 
one  minute. 

(j)  Wash  in  water,  dry,  and  mount. 

Spores  stain  red,  vegetative  cells  blue. 

2.  Capsule  Stains. — Welch  Method* — (a)  Fixed  films  of  bacteria  are 
flooded  with  glacial  acetic  acid  for  a  few  seconds. 

(6)  The  acid  is  poured  off  and  the  preparation  is  washed  two  or 
three  times  with  anilin  oil  gentian  violet,  then  flooded  with  the  stain, 
which  is  allowed  to  act  for  three  to  five  minutes. 

(c)  Wash  with  2  or  3  per  cent,  aqueous  solution  of  sodium  chloride. 

(d)  Mount  in  salt  solution  and  examine. 
Capsules  faint  purple,  bacterial  body  deep  purple. 

1  Saturated  alcoholic  solution  of  basic  fuchsin,  10  c.c.;  5  per  cent,  aqueous  phenol 
solution,  90  c.c. 

2  Cent.  f.  Bakt.,  1891,  x,  273.  3  Johns  Hopkins  Hosp.  Bull.,  1892,  128. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      181 

Hiss's  Method.1 — (a)  Place  a  drop  of  sterile  blood  serum  upon  a 
slide  and  emulsify  bacteria  in  it. 

(b)  Dry  in  the  air  and  fix  by  heat. 

(c)  Flood  smear  with  5  per  cent,   solution2  of  gentian  violet  or 
fuchsin;  steam  for  thirty  seconds. 

(d)  Remove  excess  of  stain  by  washing  in  a  20  per  cent,  solution 
of  copper  sulphate. 

(e)  Dry  with  filter  paper.    Mount  and  examine. 

Capsule  faint  pink  or  purple;  body  of  organism  deep  red  or  purple. 
Rosenow  Method.3 — (a)  Prepare  the  smear  on  perfectly  clean  cover- 
glass. 

(b)  When  smear  is  nearly  dry,  cover  with  10  per  cent,  aqueous 
solution  of  tannic  acid  for  twenty  seconds. 

(c)  Wash  with  water;  remove  moisture  with  filter  paper. 

(d)  Flood  with  anilin-oil  gentian  violet  and  steam  gently  for  thirty 
to  sixty  seconds. 

(e)  Wash  thoroughly  in  water.  ^ 

(/)  Cover  with  Gram-iodin  solution,  one  minute. 
(g)  Decolorize  with  96  per  cent,  alcohol. 

(h)  Stain  one  or  two  minutes  with  a  saturated  (60  per  cent.)  alco- 
holic solution  of  Griibler's  eosin. 

(i)  Wash  in  water;  dry  and  mount  in  balsam. 
Capsules  pink;  bacteria  blue. 
3.  Polar  Bodies. — -Neisser  Stain.4 — 
Preparation  of  Stain. — 

Solution  A — Methylene  blue 1  gram 

Ninety-six  per  cent,  alcohol    ........  20  c.c. 

Glacial  acetic  acid 50  c.c. 

Distilled  water 950  c.c. 

Solution  B — Bismarck  brown 1  gram 

Distilled  water 500  c.c. 

(a)  The  air-dried  film,  fixed  by  heat,  is  flooded  with  solution  A  for 
three  to  five  seconds. 

(b)  Wash  with  water. 

(c)  Flood  with  solution  B  for  five  seconds. 

(d)  Wash  with  water,  dry,  and  mount. 
Polar  bodies  stain  blue;  bacterial  cells  brown. 

1  Jour.  Exp.  Med.,  1905,  vi,  338. 

2  Saturated  alcoholic  solution  of  the  dye,  5  c.c.;  distilled  water,  95  c.c. 

3  Jour.  Infect.  Dis.,  1911,  ix,  1. 

4  Ztschr.  f.  Hyg.,  1897,  xxiv,  443. 


182     MICROSCOPIC  AND  CULTURAL  STUDY  OP  BACTERIA 

4.  Flagella. — Preparation  of  Film.1 — (a)  Add  enough  of  an  eighteen 
to  twenty-four-hour  agar  culture  to  a  test-tube  containing  5  c.c.  of 
sterile  salt  solution  to  produce  a  faint  turbidity  in  the  upper  half  of 
the  solution. 

(6)  Incubate  at  37°  C.  for  thirty  to  sixty  minutes. 

(c)  Place  two  or  three  loopfuls  of  the  suspension  upon  a  perfectly 
clean  cover-glass  and  allow  to  dry  spontaneously  in  the  air  or  in  the 
incubator. 

Do  not  attempt  to  spread  the  films  with  the  platinum  loop;  agita- 
tion breaks  off  flagella. 

Staining  Flagella. — Pittsfield's  Flagella  Stain. — 2 

Preparation  of  Stain. — 

(a)  Mordant: 

Tannic  acid,  10  per  cent,  aqueous  solution 10  c.c. 

Mercuric  chloride,  saturated  aqueous  solution 5  c.c. 

Alum,  saturated  aqueous  solution 5  c.c. 

Carbol  fuchsin 5  c.c. 

(b)  The  Stain: 

Alum,  saturated  aqueous  solution 10  c.c. 

Carbol  fuchsin 5  c.c.  or, 

Gentian  violet 2  c.c. 

Flood  the  dried  and  fixed  film  with  the  mordant  and  steam  gently 
for  one  minute.  Wash  in  running  water,  air-dry  and  flood  with  the 
stain.  Heat  gently  two  minutes,  wash  thoroughly  in  water,  air-dry 
and  mount. 

F.  Differential  Stains  for  Bacteria. — 1.  Gram  Stain.3 — A  most  impor- 
tant differential  method  of  staining  bacteria  is  that,  of  Gram.  Bacteria 
may  be  divided  into  two  groups:  those  which  retain  the  initial  stain 
— Gram-positive  organisms — and  those  which  fail  to  retain  the  initial 
stain  but  color  with  the  counter  stain — Gram-negative  bacteria. 

It  was  believed  formerly  that  the  organisms  which  retained  the 
initial  stain — the  Gram-positive  bacteria — contained  within  their 
protoplasm,  a  substance  of  unknown  composition  which  united 
chemically  with  gentian  violet  (or  other  pararoseanilin  dye)  and 
iodin  to  form  a  compound  relatively  insoluble  in  alcohol.  Gram- 
negative  bacteria  did  not  contain  the  hypothetical  substance,  which, 
in  association  with  the  dye  and  iodin,  was  insoluble  in  alcohol.  Treat- 
ment of  the  latter  group  with  alcohol,  therefore,  would  remove  the 

1  Kendall,  Jour.  Applied  Microscopy,  1901,  v,  1836. 

2  Medical  News,  September  7,  1895. 

3  Gram,  Fortschr.  d.  Med.,  1894,  ii, 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      183 

gentian  violet,  leaving  them  unstained.  In  the  unstained  condition 
the  organisms  were  colored  with  the  second  or  counter  stain.  Subse- 
quent investigation  has  largely  discredited  this  view.  It  has  been 
shown  by  Kruse1  that  the  cytoplasm  of  Gram-positive  bacteria  is 
more  resistant  to  autolysis,  to  the  action  of  trypsin,  and  to  solution  in 
dilute  KOH  than  that  of  Gram-negative  organisms,  probably  because 
the  cytoplasm  of  the  former  is  less  permeable  to  these  various  reagents 
than  is  that  of  the  latter.  Eisenberg,2  and  Guerbet,  Mayer  and  Schaef- 
fer3  have  advanced  the  hypothesis  that  Gram-positiveness  is  due  to 
the  lipoidal  content  of  the  cell  membrane  (ectoplasm)  and  specifically 
to  unsaturated  fatty  acids  and  phosphatids.  The  addition  of  iodin, 
according  to  this  theory,  through  the  formation  of  alcohol-insoluble 
combinations  with  the  lipoids  in  the  ectoplasm,  renders  the  cell  wall 
impermeable  to  alcohol  and  thus  prevents  removal  of  the  dye  which 
has  already  penetrated  into  the  cell  contents. 
Preparation  of  Stain: 

Solution  A — Saturated  aqueous  solution  of  anilin4 90  c.c. 

Saturated  alcoholic  solution  of  gentian  violet        .      .  10  c.c.  or, 

Five  per  cent,  aqueous  solution  of  carbolic  acid    .      .  90  c.c. 

Saturated  alcoholic  solution  of  gentian  violet        .      .  10  c.c. 

The  above  solutions  are  unstable,  but  retain  their  tinctorial  value 
for  two  or  three  days  if  they  are  kept  stoppered. 

Solution B5 — Distilled  water 300  c.c. 

Potassium  iodide 2  grams 

Iodin  crystals 1  gram 

Solution  C — Bismarck  brown,  saturated  aqueous  solution  ...        10  c.c. 
Distilled  water 90  c.c. 

Procedure. — (a)  Prepare  and  fix  film  of  bacteria  in  the  usual  manner. 
(6)  Flood  with  anilin-oil  gentian  violet  (or  carbolic  gentian  violet) 
and  stain  five  minutes. 

(c)  Pour  off  excess  of  stain  and  flood  with  iodin  solution. 

(d)  Decolorize  with  96  per  cent,  alcohol  until  no  more  stain  can  be 
removed. 

(e)  Wash  thoroughly  in  water. 

(/)  Counterstain  with  Bismarck1  brown6 13r  two  minutes. 
(g)  Wash  in  water,  dry,  and  mount. 

1  Miinchen.  med.  Wchnschr.,  1910,  p.  685. 

2  Cent.  f.  Bakt.,  1909,  xlix,  465;  1910,  li,  115;  liii,  481,  551;  Ivi,  183. 

3  Compt.  rend.,  Soc.  biol.,  Ixviii,  353. 

4  Three  c.c.  of  anilin  oil  are  shaken  for  several  minutes  in  100  c.c.  of  distilled  water. 
The  solution  is  filtered  through  filter  paper  to  remove  the  undissolved  anilin. 

5  This  iodin  solution  is  variously  known  as  Gram's  iodin  solution  or  Lugol's  solution. 

6  Dilute  aqueous  fuchsin,  1  to  10,  may  be  used  in  place  of  Bismarck  brown. 


184     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

Bacteria  which  retain  the  initial  stain — Gram-positive  bacteria — 
are  colored  dark  purple  or  blue;  those  which  fail  to  retain  the  initial 
stain— Gram-negative  bacteria — are  brown,  or  bright  pink  if  fuchsin 
is  used  as  a  counterstain. 

2.  Stains  for  Acid-fast  Bacteria. — Ziehl-Neelsen  Method.1 — (a)  Stain 
dried  and  fixed  smear  with  carbol  fuchsin,  as  described  on  page  180. 

(6)  Wash  thoroughly  with  water. 

(c)  Decolorize  with  acid  alcohol2  until  no  more  color  can  be  washed 
out. 

(d)  Wash  with  water. 

(e)  Counterstain  lightly  with  Loffler's  alkaline  methylene  blue. 
(/)  Wash,  dry,  and  mount. 

Acid-fast  bacilli  and  spores  red;  all  others  blue. 

3.  Frdnkel-Gabbet  Method.3 — (a)  Stain  with  carbol  fuchsin  as  in 
the  Ziehl-Neelsen  method  and  wash  in  water. 

(b)  Decolorize  and  counterstain  simultaneously  with  the  following 
solution : 

Methylene  blue 2  grams 

Water .     75  c.c. 

Sulphuric  acid 25  c.c. 

The  counterstain  is  allowed  to  act  for  one  minute. 

(c)  Wash,  dry,  and  mount. 

4.  Polychrome  Stains. — Polychrome  stains  are  of  special  value  for 
the  examination  of  exudates,  body  fluids  or  tissues  in  which  the  his- 
tological  relations  of  bacteria  are  to  be  investigated.    These  stains, 
or  modifications  of  them,  are  also  useful  in  the  study  of  treponemata, 
spirochetes,  and  protozoa. 

Wright's  Stain.4 — Preparation. — "To  a  0.5  per  cent,  aqueous 
solution  of  sodium  bicarbonate  add  methylene  blue  (B.X.,  or  '  medi- 
cinally pure')  in  the  proportion  of  1  gm.  of  the  dye  to  each  100  c.c. 
of  the  solution.  Heat  the  mixture  in  a  steam  sterilizer  at  100°  C.  for 
one  full  hour,  counting  the  time  after  the  sterilizer  has  become  thor- 
oughly heated.  The  mixture  is  to  be  contained  in  a  flask,  or  flasks, 
of  such  size  and  shape  that  it  forms  a  layer  not  more  than  6  cm.  deep. 
After  heating  the  mixture  is  allowed  to  cool,  placing  the  flask  in  cold 
water  if  desired,  and  is  then  filtered  to  remove  the  precipitate  which 

1  Ziehl,  Deutsch.  med.  Wchnschr.,  1882,  451;  Neelsen,  Fort.  d.  med.,  1885,  200. 

2  Ninety  per  cent,  alcohol  containing  3  per  cent,  by  volume  of  hydrochloric  acid. 

3  Frankel,  Berl.  klin.  Wchnschr.,  1884;  Gabbet,  Lancet,  1887. 

4  Mallory  and  Wright,  Pathological  Technic,  1915,  6th  ed.,  p.  382. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      185 

has  formed  in  it.  It  should  when  cold  have  a  deep  purple  red  color 
when  viewed  in  a  thin  layer  by  transmitted  yellowish  artificial  light. 
It  does  not  show  this  color  while  it  is  warm. 

"To  each  100  c.c.  of  the  filtered  mixture  add  500  c.c.  of  a  0.1  per 
cent,  aqueous  solution  of  'yellowish,  water-soluble'  eosin  and  mix 
thoroughly.  Collect  on  a  filter  the  abundant  precipitate  which  imme- 
diately appears.  When  the  precipitate  is  dry,  dissolve  it  in  methylic 
alcohol  (Merck's  'reagent')  in  the  proportion  of  0.1  gm.  to  60  c.c. 
of  the  alcohol.  In  order  to  facilitate  solution  the  precipitate  is  to  be 
rubbed  up  with  alcohol  in  a  porcelain  dish  or  mortar  with  a  spatula 
or  pestle. 

"This  alcoholic  solution  of  the  precipitate  is  the  staining  fluid.  It 
should  be  kept  in  a  well-stoppered  bottle  because  of  the  volatility  of 
the  alcohol.  If  it  becomes  too  concentrated  by  evaporation  and  thus 
stains  too  deeply,  or  forms  a  precipitate  on  the  blood  smear,  the 
addition  of  a  suitable  quantity  of  methylic  alcohol  will  quickly  correct 
such  faults.  It  does  not  undergo  any  other  spontaneous  change  than 
that  of  concentration  by  evaporation. 

"A  most  important  fault  met  with  in  the  working  of  some  samples 
of  this  fluid  is  that  it  fails  to  stain  the  red  blood  corpuscles  a  yellow  or 
orange  color,  but  stains  them  a  blue  color  which  cannot  readily  be 
removed  by  washing  with  water.  This  fault  is  due  to  a  defect  in  the 
specimen  of  eosin  employed.  It  can  be  eliminated  by  using  a  proper 
'yellowish,  water-soluble'  eosin." 

Method  of  Staining. — (a)  Unheated  air-dried  films1  are  covered 
with  the  stain,  which  is  allowed  to  act  for  one  minute. 

(6)  Add  an  equal  volume  of  distilled  water  to  the  stain  and  allow  to 
stand  for  three  minutes. 

(c)  Wash  in  water  for  thirty  seconds,  or  until  a  pink  color  develops. 

(d)  Dry  rapidly  with  filter  paper  and  mount  in  balsam.2 
Giemsa  Method.3 — Preparation  of  Stain: 

Azur  II  (eosin) 3.0  grams 

Azur  II 0.8  grams 

Glycerin,  C.  P 250  c.c. 

Neutral  absolute  methyl  alcohol 250  c.c. 

The  dyes  are  dissolved  in  the  glycerin  at  60°  C.;  the  alcohol,  warmed 
to  40°  C.,  is  then  added,  thoroughly  mixed  by  shaking,  and  allowed 
to  cool  slowly  to  room  temperature,  then  filtered.  Immediately  before 

1  Films  more  than  a  few  hours  old  do  not  stain  as  well  as  fresh  ones. 

2  The  balsam  must  be  neutral  in  reaction. 

3  Giemsa,  Cent.  f.  Bakt.,  1904,  xxxvii,  308. 


186     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

use,  10  c.c.  of  distilled  water  are  slightly  alkalinized  by  the  addition 
of  two  drops  of  a  10  per  cent,  solution  of  sodium  carbonate,  and 
exactly  ten  drops  of  the  stain  are  then  added. 

Staining  with  Giemsa  Solutions. — (a)  Films  are  fixed  by  immersion 
in  neutral  absolute  methyl  alcohol  for  one  minute,  air-dried,  and 
covered  with  the  diluted  stain,  which  is  allowed  to  act  for  fifteen  to 
twenty  minutes  when  ordinary  exudates  and  bacteria  are  used;  for 
one  to  three  hours  if  Treponemata  or  Negri  bodies  are  sought  for. 

(b)  Wash  in  water,  dry  and  mount. 

5.  W.  H.  Smith's  Solution  Stain. — (a)  Stain  the  fixed  smear  with 
anilin  oil  gentian  violet  for  one  minute. 

(b)  Wash  with  water. 

(c)  Flood  with  Gram-iodin  solution  for  thirty  seconds. 

(d)  Decolorize  with  95  per  cent,  alcohol. 

(e)  Wash  with  ether  for  a  few  seconds. 

(/)  Flood  with  absolute  alcohol  for  five  seconds. 

(g)  Stain  with  saturated  aqueous  solution  eosin  for  one  to  two 
minutes. 

(k)  Wash  with  absolute  alcohol  for  a  few  seconds. 

(i)  Clear  with  xylol. 

( j)  Mount  in  balsam. 

III.  Staining  Bacteria  in  Tissues. — Paraffin  sections  are  preferable, 
partly  because  very  thin  sections  may  be  cut;  chiefly  because  celloidin 
stains  somewhat  with  the  stains  ordinarily  used. 

The  Gram-Weigert  Stain  for  Bacteria  in  Tissues.1 — (a)  Stain  paraffin 
sections  with  anilin  oil  methyl  violet  for  five  to  twenty  minutes. 

(6)  Wash  in  water  to  remove  excess  of  stain. 

(c)  Gram-iodin  solution  for  one  minute. 

(d)  Wash  in  water  to  remove  excess  of  iodin. 

(e)  Decolorize  with  several  changes  of  absolute  alcohol  until  no 
more  color  comes  out. 

(/)  Clear  section  in  xylol. 
(g)  Mount  in  neutral  xylol  balsam. 

Mallory  and  Wright  Modification  for  Celloidin  Sections.2 — (a)  Stain 
sections  with  lithium  carmine  for  two  to  five  minutes. 

(b)  Remove  excess  of  stain  with  acid  alcohol. 

(c)  Wash  in  water. 

(d)  Dehydrate  in  95  per  cent,  alcohol. 

1  Mallory  and  Wright,  Pathological  Technic,  6th  ed.,  1915,  p.  432. 
*  Ibid. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      187 

(e)  Expose  to  ether  vapor  to  fix  section  to  slide. 
(/)  Stain  with  anilin  oil  methyl  violet  for  five  to  twenty  minutes. 
(g)  Remove  excess  stain  with  normal  salt  solution. 
(h)  Gram-iodin  solution  for  one  minute. 
(i)  Remove  excess  iodin  with  water. 

( j)  Remove  moisture  as  thoroughly  as  possible  with  filter  paper. 
(k)  Dehydrate  in  several  changes  of  anilin  oil. 
(/)  Clear  with  several  changes  of  xylol. 
(m)  Mount  in  neutral  xylol  balsam. 

Staining  Tubercle  Bacilli  in  Tissues. — (a)  Paraffin  sections  are 
covered  with  carbol-fuchsin  and  steamed  gently  for  five  minutes. 

(b)  The  excess  stain  is  removed  with  water. 

(c)  Decolorize  and  counterstain  with  Gabbet  methylene-blue  sul- 
phuric acid  stain  about  one  minute. 

(d)  Remove  excess  of  stain  and  acid  with  water. 

(e)  Dehydrate  with  absolute  alcohol. 
(/)  Clear  section  in  xylol. 

(g)  Mount  in  xylol  balsam. 

Staining    Actinomyces    in    Tissues — Mallory    Method.1 — (a)  Stain 
paraffin  sections  with  saturated  aqueous  eosin  for  ten  minutes. 
(6)  Remove  excess  stain  with  water. 

(c)  Stain  with  anilin  oil  methyl  violet  for  two  to  five  minutes. 

(d)  Remove  excess  stain  with  normal  salt  solution. 

(e)  Remove  excess  water  with  filter  paper. 
(/)  Clear  in  anilin  oil. 

(g)  Remove  anilin  oil  with  several  changes  of  xylol. 

(h)  Mount  in  neutral  xylol  balsam. 

The  clubs  stain  pink,  the  filaments  blue. 

IV.  Methods  and  Media  for  the  Cultivation  of  Bacteria. — One  of 
the  most  important  procedures  in  bacteriology  is  the  preparation 
of  nutritive  media  in  which  the  morphology,  chemistry,  and  cultural 
characteristics  of  the  organism  may  be  studied;  furthermore,  it  is 
possible  by  cultural  methods  to  separate  one  type  of  bacterium  in 
pure  culture  from  associated  organisms,  and  to  study  its  reactions 
apart  from  all  contaminating  microorganisms.  The  technic  of  isolat- 
ing and  cultivating  bacteria  is  exacting  at  every  step  of  the  process, 
from  the  preparation  of  glassware  to  the  selection  of  suitable  nutritive 
media,  and  their  preparation  requires  not  only  scrupulous  cleanliness; 
it  necessitates  a  most  rigorous  maintenance  of  sterility. 

1  Loc.  cit.,  p.  433. 


188     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

Bacterial  cultivation  is  usually  carried  out  in  glass  vessels — test- 
tubes,  flasks,  fermentation  tubes,  and  Petri  dishes — because  glass  is 
transparent  and  permits  an  unobstructed  view  of  the  reactions  taking 
place  within.  It  is  obvious  that  glassware  employed  in  bacterial 
laboratories  must  be  chemically  and  bacteriologically  clean. 

Preparation  of  Glassware. — The  method  to  be  employed  in  the  clean- 
ing of  glassware  depends  somewhat  on  the  purpose  for  which  it  is 


FIG.  12.— Petri  dish. 

used.  New  glassware  frequently  contains  alkali,  which  is  readily 
neutralized  by  diluted  acid,  hydrochloric  or  sulphuric.  Glassware 
that  has  contained  cultures  of  bacteria  is  first  sterilized  in  the  auto- 
clave to  remove  all  danger  of  infection,  then  immersed  in  a  strong 
solution  of  soap-powder  and  soap-suds  maintained  at  a  boiling  tem- 
perature for  half  an  hour.  The  adherent  media  is  removed  with  a 
brush  or  swab;  a  final  thorough  rinsing  in  clear  water  removes  all 


FIG.  13. — Fermentation  tubes — various  types. 

traces  of  soap.  Very  dirty  glassware  or  glassware  in  which  chemical 
determinations  are  to  be  made  should  be  cleaned  in  chromic  acid 
solution,  which  is  prepared  by  adding  a  saturated  aqueous  solution 
of  potassium  bichromate  to  a  1  to  3  dilution  of  sulphuric  acid.  Twenty- 
four  hours'  exposure  to  chromic  acid  removes  all  traces  of  organic 
matter,  as  a  rule.  Following  the  acid  bath  the  glassware  is  thoroughly 
rinsed  in  clear  water  and  dried. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      189 

The  cleaned  glassware — test-tubes,  flasks,  or  fermentation  tubes — 
is  then  stoppered  with  non-absorbent  cotton — cotton  batting — which 
has  a  long  staple  or  fiber.  The  cotton  plugs  must  be  carefully  fitted — 
neither  too  loose,  which  would  permit  of  the  passage  of  adventitious 
microorganisms,  nor  too  tight,  for  obvious  reasons.  The  cotton  plugs 
are  conveniently  prepared  from  a  layer  of  cotton  batting  about  two 
inches  square  (for  a  test-tube  of  ordinary  diameter,  about  15  mm.), 
which  is  laid  squarely  over  the  orifice.  The  center  of  the  square  is 
gently  pushed  down  into  the  neck  of  the  tube  for  a  distance  of  about 
three-fourths  to  one  inch;  sufficient  cotton  protrudes  from  the  tube 
to  be  conveniently  grasped  by  the  fingers  and  removed.  It  is  fre- 
quently advisable  to  cover  the  cotton  plugs  with  two  or  three  layers 
of  filter  paper,  which  prevents  an  accumulation  of  dust  on  the  cotton. 
Wide-mouthed  containers  are  sealed  with  several  layers  of  unglazed 
paper  fastened  in  place  with  a  piece  of  twine.  Flasks  are  frequently 
not  plugged  with  cotton;  the  neck  is  simply  covered  by  an  inverted 
beaker  of  appropriate  size. 

Glassware  should  always  be  sterilized  before  media  is  placed  in 
it;  this  is  readily  accomplished  by  dry  heat.  A  hot-air  sterilizer  is 
used,  in  which  a  temperature  of  180°  C.  is  maintained  for  one  hour. 
A  higher  temperature  must  be  avoided,  to  prevent  charring  of  cotton 
plugs.  The  heat  must  be  increased  gradually  and  diminished  gradually, 
to  prevent  cracking  of  the  glass.  By  this  process  not  only  is  the 
utensil  rendered  sterile,  the  plugs  of  cotton  retain  their  shape  when 
withdrawn,  as  well. 

A  majority  of  the  bacteria  pathogenic  for  man  and  many  parasitic 
and  saprophytic  forms  as  well  require  relatively  complex  organic 
compounds  containing  carbon,  hydrogen,  nitrogen,  and  oxygen, 
together  with  other  elements  for  their  nutrition.  These  foodstuffs 
provide  both  the  structural  and  fuel  requirements  of  the  organism, 
as  explained  in  the  chapter  on  Bacterial  Metabolism.  Experience  has 
shown  that  a  medium  containing  meat  infusion,  peptone,  and  salt  is 
a  satisfactory  one  for  many  bacteria.  This  medium  may  be  enriched 
by  the  addition  of  various  ingredients  to  meet  the  requirements  of  the 
more  fastidious  organisms. 

Meat  infusion  is  prepared  from  finely  comminuted  lean  meat1  freed 
from  fat.  500  grams  of  meat  are  intimately  mixed  with  1000  c.c.  water 
and  allowed  to  infuse  over  night  in  the  refrigerator.  It  is  then  strained 

1  Beef  hearts  make  a  very  satisfactory  meat  infusion  and  their  cost  is  much  less  than 
the  better  cuts  of  meat. 


190     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

through  several  layers  of  cheese-cloth,  the  volume  recorded,  then 
heated  to  boiling.  The  coagulum  which  forms  is  removed  by  filtration 
through  filter  paper  and  the  clear,  amber-colored  fluid,  after  restoring 
the  loss  due  to  evaporation,  is  run  into  flasks  and  sterilized  in  an 
autoclave  at  15  pounds'  pressure  for  fifteen  minutes.  This  plain  meat 
infusion  contains  but  little  protein;  it  is  relatively  rich,  however,  in 
soluble  meat  extractives,  soluble  salts  and  muscle-sugar — dextrose. 
It  is  not  suitable  in  itself  as  a  complete  nutritive  medium  for  most 
bacteria,  but  it  forms  the  basis  of  many  of  the  commonly  used  nutri- 
tive media.  Meat  extract  (Liebig's  or  other  kinds)  is  frequently 
substituted  for  meat  infusion.  Three  grams  of  meat  extract  are  dis- 
solved in  a  small  volume  of  water,  filtered  through  a  cold  wet  filter 
paper  to  remove  fat,  and  made  up  to  a  volume  of  a  liter.  The  solution 
contains  some  meat  extractives,  including  a  relatively  large  propor- 
tion of  xanthin  bases  and  a  very  considerable  amount  of  salts,  par- 
ticularly sodium  chloride.  Little  or  no  muscle-sugar  is  present.  It 
is  distinctly  inferior  to  meat  infusion,  however,  as  a  basis  for  cultural 
media,  especially  for  the  more  delicate  pathogenic  organisms. 

The  Reaction  of  Media. — Bacteria  are  relatively  sensitive  to  com- 
paratively slight  changes  in  the  reaction  of  their  nutritional  environ- 
ment, and  it  is  essential  to  create  a  suitable  initial  degree  of  acidity 
or  alkalinity  in  media  to  favor  their  growth.  A  reaction  neutral  to 
phenolphthalein — slightly  alkaline  to  litmus — is  suitable  for  most  of 
the  bacteria  pathogenic  for  man — human  tissues  and  blood  are  slightly 
alkaline  to  litmus.  A  reaction  of  1  per  cent,  acid  (+1.0),  using  phenol- 
phthalein as  an  indicator,  has  been  recommended  by  the  Laboratory 
Section  of  the  American  Public  Health  Association  for  the  routine 
bacterial  examination  of  water,  ice,  sewage,  milk,  cream,  and  ice-cream. 
A  reaction  of  1  per  cent,  signifies  that  1  c.c.  of  normal  NaOH  would 
be  required  to  neutralize  the  acid  in  100  c.c.  of  the  medium.  Ten 
c.c.  of  Y  NaOH  would  be  required  to  exactly  neutralize  one  liter  of 
medium  having  an  acidity  of  1  per  cent. 

The  reaction  may  be  determined  accurately  in  the  following  manner : 
to  45  c.c.  of  distilled  water,  contained  in  a  porcelain  evaporating  dish 
of  100  c.c.  capacity,  are  delivered  exactly  5  c.c.  of  the  medium  from  a 
graduated  pipette.  The  solution  is  brought  to  the  boiling-point  over 
the  free  flame  to  expel  CO2  and  1  c.c.  of  a  solution  of  phenolphthalein1 

1  Made  by  dissolving  0.5  gram  phenolphthalein  in  100  c.c.  50  per  cent,  alcohol.  This 
indicator  is  colorless  in  acid  solution — pink  in  an  alkaline  solution.  CO2  interferes  with 
its  accuracy  as  an  indicator.  It  is  especially  sensitive  to  organic  acids  which  occur  in 
ordinary  media,  hence  its  value  in  media  titrations. 


METHODS  FOR   THE  MICROSCOPIC  STUDY  OF  BACTERIA      J91 

is  added  as  an  indicator.  The  solution  usually  remains  colorless, 
because  ordinary  media  are  acid  in  reaction;  Jg  NaOH  is  added  slowly 
from  a  burette  until  a  faint  pink  color  appears  and  persists  after  one 
minute's  boiling.  From  the  amount  of  f0  alkali  required  to  neutralize 
5  c.c.  of  medium,  the  reaction  of  the  entire  amount  is  readily  com- 
puted. Thus: 

5  c.c.  media  are  neutralized  by  3  c.c.  ^  NaOH. 
100  c.c.  media  would  be  neutralized  by  3  c.c.  normal  (y)NaOH. 
1000  c.c.  media  would  be  neutralized  by  30  c.c.  normal  (j)NaOH. 

To  reduce  the  reaction  of  a  liter  of  medium  whose  initial  reaction 
is  +  3.0  to  +  1.0,  20  c.c.  of  normal  NaOH  would  be  required.  It  is 
necessary  to  heat  the  medium  after  adding  the  alkali,  in  order  to 
promote  the  reaction  between  the  acids  of  the  medium  and  the  neu- 
tralizing solution  and  a  redetermination  of  the  reaction  should  be 
made  to  make  certain  that  the  desired  change  in  acidity  has  taken 
place.  Frequently  a  second  addition  of  alkali  is  necessary  to  create 
the  desired  final  reaction. 

A  satisfactory  reaction  for  cultural  media  designed  for  most  patho- 
genic bacteria  may  be  created  by  adding  ^  NaOH  solution— a  few 
drops  at  a  time,  to  the  entire  volume,  using  filter  paper  dipped  in 
phenolphthalein  solution,  and  dried,  as  an  indicator.  When  the  paper 
shows  a  faint  pink  color  the  addition  of  alkali  is  discontinued.  The 
reaction  is  practically  neutral  under  these  conditions. 

The  Clarification  of  Media. — It  is  desirable,  in  the  preparation  of 
culture  media,  to  remove  all  insoluble  substances.  This  is  accom- 
plished by  filtration  methods,  with  or  without  preliminary  treatment, 
to  flocculate  the  substances  in  suspension.  The  addition  of  non-heat- 
coagulable  proteins,  as  gelatin,  frequently  requires  clarification  with 
a  coagulable  protein,  as  egg-albumen,  to  remove  the  finely  divided 
suspended  matter. 

Clarifying  with  Eggs. — For  each  liter  of  medium  to  be  clarified,  two 
eggs  thoroughly  whipped  in  a  small  amount  of  water  are  added.  The 
temperature  of  the  medium  should  not  exceed  50°  C.  The  eggs  are 
thoroughly  stirred  in  and  the  entire  mixture  is  slowly  heated  to  100° 
C.,  either  in  a  double  boiler  or  in  the  Arnold  sterilizer.  A  firm  coagulum 
forms  during  the  heating  process,  which  enmeshes  the  suspended  par- 
ticles it  is  desired  to  remove.  The  medium  should  never  be  disturbed 
during  the  coagulating  process.  The  clear  underlying  medium  is 
drawn  off  and  filtered  through  cotton. 


192     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

Filtration  through  Cotton. — A  large  glass  funnel  is  lined  with  a  double 
layer  of  absorbent  cotton;  the  layers  are  placed  at  right  angles, 
thus  laying  the  fibers  of  cotton  at  right  angles.  The  cotton  is  moistened 
with  a  small  amount  of  hot  water  if  agar  or  gelatin  is  to  be  filtered. 
The  medium  is  then  carefully  poured  into  the  funnel,  care  being  taken 
that  the  cotton  is  not  displaced  by  the  force  of  the  inflowing  fluid.  The 
first  portion  of  filtrate  may  not  be  clear  and  it  is  somewhat  diluted 


FIG.  14. — Hot-air  sterilizer.     Lautenschlager  form.     (Park.) 

with  the  water  originally  used  to  wet  the  cotton — hence  it  should  be 
returned  and  refiltered.  Agar  and  gelatin  filter  slowly,  which  may 
lead  to  congelation,  therefore  the  top  of  the  funnel  should  be  covered 
to  prevent  undue  loss  of  heat.  Funnels  surrounded  by  a  hot  water 
jacket  are  sometimes  used  in  the  filtration  of  these  media.  Media 
that  are  fluid  when  cold  may  be  often  advantageously  clarified  by 
filtration  through  a  good  grade  of  heavy  filter  paper,  with  or  without 
a  preliminary  clarification  with  eggs,  as  occasion  demands. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      193 


The  Distribution  of  Media. — The  clarified  media  are  either  stored  in 
flasks  or  transferred  to  smaller  containers  for  immediate  use.  Then 
they  are  sterilized.  Most  media  are  used  in  test-tubes.  Test-tubes 
are  filled  from  a  reservoir,  usually  a  large  funnel,  the  smaller  end  of 
which  is  provided  with  a  short  length  of  rubber  tubing,  into  which  a 
short  glass  tube,  constricted  somewhat  at  the  outer  end,  is  introduced. 
The  flow  is  controlled  by  a  pinch  cock,  which  constricts  the  rubber 
tubing  midway  between  the  funnel  and  the  delivery  tube.  The  cotton 
plug  is  removed  from  a  test-tube  and  the  delivery  tube  is  introduced 
into  the  open  end  of  it  to  a  depth  of  about  two  inches.  The  pinch 


FIG.  15. — Arnold  steam  sterilizer. 
(Abbott.) 


STERILIZING  CHAMBER 

U          A      4  A 


FIG.  16. — Arnold  steam  sterilizer. 
Ordinary  type.     (Park.) 


cock  is  opened  somewhat  and  the  desired  volume  is  allowed  to  flow  in. 
The  pinch  cock  is  then  released  to  stop  the  flow,  the  delivery  tube 
removed,  care  being  taken  that  no  media  touches  that  part  of  the 
test-tube  where  the  cotton  fits,  so  that  it  will  not  adhere  to  the  sides 
of  the  tube,  and  the  cotton  plug  is  replaced.  Usually  about  8  to  10 
c.c.  of  media  are  added  to  a  tube. 

Sterilization  of  Media. — Media  which  do  not  contain  coagulable 
proteins,  gelatin  or  carbohydrates  are  sterilized  for  fifteen  minutes  in 
an  autoclave  at  a  live  steam  pressure  of  fifteen  pounds  (121.3°  C.). 
Media  containing  gelatin  or  carbohydrates  are  sterilized  at  a  lower 
temperature  by  discontinuous  sterilization — half  an  hour  on  three 

13 


194     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

successive  days,  in  flowing  steam  in  an  Arnold  sterilizer.  After  each 
sterilization  the  medium  is  kept  at  room  temperature  to  permit  of  the 
germination  of  spores.  Lower  temperatures  are  occasionally  employed, 
particularly  for  the  sterilization  of  blood  serum  or  other  native  pro- 
teins— an  exposure  of  to  70°  C.  for  an  hour  on  each  of  six  successive 
days  usually  suffices.  Bacteria  may  be  removed  from  fluid  media 
and  from  various  sera  and  solutions  containing  thermolabile  toxins 
or  similar  products  by  filtration  through  sterile  porous  filters  made  of 
unglazed  porcelain  or  diatomaceous  earth — Pasteur  or  Berkefeld 
filters.  These  filters  are  made  with  varying  degrees  of  porosity, 


FIG.  17. — Arnold  steam  sterilizer.     Boston  Board  of  Health  type.     (Park.) 

regulated  largely  by  the  thickness  of  their  walls  to  accommodate  vary- 
ing needs.  Usually  the  fluid  is  forced  through  the  walls  of  the  filter 
into  the  center,  which  is  hollow,  by  suction.  The  clear,  bacteria-free 
filtrate  passes  into  a  sterile  container  attached  to  the  filter.  The  filters 
and  their  necessary  accessory  parts  are  sterilized  in  the  autoclave  for 
fifteen  minutes  at  fifteen  pounds  live-steam  pressure.  Turbid  fluids 
should  be  passed  through  several  layers  of  filter  paper  prior  to  filtra- 
tion, to  remove  the  grosser  particles  which  otherwise  would  soon 
clog  the  filter.  A  time  limit,  usually  not  exceeding  two  hours  as  a 
maximum,  should  be  set,  beyond  which  filtration  should  be  stopped 


METHODS  FOR   THE  MICROSCOPIC  STUDY  OF  BACTERIA      195 

—bacteria  may  be  forced  through  filters  and  contaminate  the  filtrate 
if  the  process  is  carried  much  beyond  this  interval. 

New,  unused  filters  should  be  cleaned  by  running  several  liters  of 
clean  water  through  them  and  they  should  invariably  be  tested  before 
use  to  guard  against  "  pin-holes." 

After  filtration  the  filter  is  sterilized  to  kill  whatever  bacteria  have 
contaminated  it.  Then  the  surface  is  thoroughly  scrubbed  with  a 
brush  and  1  per  cent,  alkaline  permanganate  solution  (potassium  per- 
manganate 10  grams,  water  1000  c.c.)  is  run  through  to  remove  organic 
matter.  Five  per  cent,  oxalic  acid  is  then  passed  through  to  remove 


FIG.  91  FIG.  92  FIG.  93  FIG.  94 

FIGS.  91  to  94. — Types  of  unglazed  porcelain  filters.     (Park.) 


the  permanganate  solution  and  the  acid  finally  removed  by  repeated 
washings  with  water.  If  the  filter  becomes  so  clogged  with  organic 
matter  that  it  can  no  longer  deliver  a  reasonable  amount  of  filtrate, 
the  filter  is  placed  in  a  muffle-furnace,  gradually  heated  to  about 
250°  C.,  and  as  gradually  cooled.  It  is  then  cleaned  as  before  with 
permanganate  solution,  to  remove  the  last  traces  of  organic  matter. 

Storage  of  Media. — If  media  are  not  to  be  used  at  once  it  is  necessary 
to  protect  them  from  evaporation  and  contamination.  Flasks  of  media 
are  preserved  best  by  tying  paper  caps  over  the  cotton  plugs  if  the 
period  of  storage  does  not  exceed  a  few  days,  or  by  pouring  melted 
paraffin  over  the  plugs  if  longer  periods  of  storage  are  contemplated. 


196     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

It  is  necessary  to  burn  the  surface  of  the  plug  to  destroy  surface  con- 
tamination, then  to  push  the  plug  into  the  neck  of  the  flask  for  a  dis- 
tance of  1  cm.  to  make  room  for  the  paraffin.  Flasks  hermetically 
sealed  in  this  manner  may  remain  visibly  unchanged  for  weeks  or  even 
months.  It  is  good  practice  to  place  a  lead  foil  cap  over  the  paraffin 


FIG.  22.— Autoclave.     (Park.) 


plug  and  lead  foil  caps  are  better  than  paper  caps  as  coverings  for  cot- 
ton plugs.  Media  in  storage  should  be  maintained  at  a  temperature 
not  exceeding  45°  C.,  in  a  dry  ice-box. 

The  Preparation  of  Nutrient  Bouillon  (Broth).— M eat  Infusion  Broth  — 
To  1000  c,c.  of  meat  infusion  (see  page  189  for  preparation),  in  a  tared, 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      197 

agate-ware  boiler,  add  5  grams  of  common  salt  (NaCl)  and  heat  to 
boiling.  Dust  10  grams  of  Witte  peptone  over  the  surface  and  stir 
until  it  is  thoroughly  dissolved.  Restore  the  loss  by  evaporation  and 
adjust  the  reaction  to  the  desired  degree  of  acidity.  Boil  for  five 
minutes,  verify  the  reaction  and  filter  through  filter  paper  until  the 
filtrate  is  perfectly  clear.  Sterilize  in  the  autoclave. 

Meat  Extract  Broth.1— To  1000  c.c.  of  meat  extract  (see  page  190  for 
preparation)  in  a  tared  agate-ware  boiler,  add  10  grams  of  Witte  pep- 
tone, dusting  the  peptone  on  the  surface.  Heat  to  boiling,  restore 
loss  by  evaporation  and  adjust  the  reaction.  Continue  the  boiling  for 
five  minutes,  verify  the  reaction  and  cool  to  room  temperature.2  Filter 
cold  through  filter  paper  until  perfectly  clear  and  sterilize. 

Nutrient  Sugar-free  Broth. — Meat  infusion  contains  small  amounts 
of  muscle-sugar — dextrose — usually  about  0.1  per  cent.  This  sugar 
is  present  in  nutrient  meat  infusion  broth  prepared  as  outlined  above. 
It  is  frequently  desirable  to  prepare  meat  infusion  broth  free  from  all 
sugars.  The  dextrose  is  readily  removed  by  fermentation  with  Bacillus 
coli,  adding  a  broth  culture  of  this  organism  to  the  meat  infusion  before 
it  is  heated  and  maintaining  the  infusion  at  37°  C.,  for  eighteen  to 
twenty-four  hours.  The  sugar  which  is  attacked  by  Bacillus  coli  in 
preference  to  the  protein  constituents  of  the  medium3  is  quantitatively 
removed.  The  organism  must  be  killed  as  soon  as  the  sugar  is 
exhausted,  otherwise  the  protein  constituents  will  be  attacked.  The 
end  of  the  fermentation  may  be  judged  with  a  fair  degree  of  certainty 
if  one  removes  some  of  the  infusion  seeded  with  Bacillus  coli  to  a  fer- 
mentation tube,  kept  at  the  same  temperature,  37°  C.;  when  gas  is 
no  longer  evolved  the  sugar  is  exhausted.  Sugar-free  broth  contains 
lactic  acid,  one  of  the  products  of  fermentation  of  dextrose  by  Bacillus 
coli.  After  the  sugar  is  removed  the  medium  is  sterilized  in  the  usual 
manner,  or  made  directly  into  sugar-free  nutrient  meat  infusion  broth 
as  outlined  above. 

Nutrient  Sugar  Broth. — One  per  cent,  of  dextrose,  lactose,  saccharose, 
mannite,  or  other  carbohydrate  is  added  to  nutrient  sugar-free  broth 
immediately  before  filtering.  Media  containing  sugars  are  best  steri- 
lized in  the  Arnold  sterilizer  on  three  successive  days;  the  high  tem- 
perature of  the  autoclave  tends  to  decompose  carbohydrates. 

1  It  is  unnecessary  to  add  salt  to  meat  extract. 

2  A  precipitate  containing  phosphates,  soluble  in  the  hot  medium,  settles  out  upon 
cooling.    It  must  be  removed  before  the  medium  is  used. 

8  See  chapter  on  Bacterial  Metabolism. 


198     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

Calcium  Carbonate  Nutrient  Sugar  Media. — Bacteria  grown  in 
sugar  media  frequently  form  acid  products  from  the  fermentation  of 
the  sugars — the  amount  of  acid  products  may  be  sufficient  to  inhibit 
the  development  of  the  organisms  even  after  one  or  two  days' 
growth.  The  addition  of  insoluble  carbonates — as  calcium  carbonate 
— neutralizes  the  acid  as  it  is  formed  and  thus  maintains  automatically 
a  favorable  reaction  for  prolonged  development.  Bolduan1  has  shown 
that  pieces  of  marble  about  0.5  centimeters  square  in  100  c.c.  of  broth 
not  only  restrain  the  development  of  free  acid — the  marble  appears  to 
create  a  somewhat  more  favorable  medium,  especially  for  the  pneumo- 
coccus  and  streptococcus  as  well.  The  bits  of  marble  should  be- 
sterilized  in  the  hot-air  sterilizer  before  they  are  introduced  into  the 
broth. 

Nutrient  Glycerin  Broth. — To  1  liter  of  sugar-free  broth  add  3  to 
5  per  cent,  pure,  redistilled  glycerin  immediately  before  filtering. 
Sterilize  in  the  autoclave  fifteen  minutes  at  fifteen  pounds  pressure. 
Glycerin  broth  is  extensively  used  for  the  cultivation  of  the  tubercle 
bacillus2  and  it  is  frequently  employed  in  the  culture  of  bacteria  which 
are  susceptible  to  desiccation — the  glycerin  conserves  the  moisture 
and  retards  evaporation. 

The  various  sugar-broths  may  be  prepared  with  meat  extract  as 
a  basis;  pathogenic  bacteria  develop  less  luxuriantly  as  a  rule  in 
extract  media  than  in  meat  infusion  media,  however. 

Dunham's  Solution. — Five  grams  of  common  salt  and  10  grams  of 
Witte  peptone  are  added  to  one  liter  of  water  and  heated  to  boiling 
until  the  peptone  is  completely  dissolved.  Pass  through  filter  paper 
until  perfectly  clear,  tube,  using  10-  c.c.  to  each  test  tube,  and  sterilize 
in  the  autoclave.  The  reaction  does  not  require  adjustment. 

This  medium  is  frequently  used  to  test  the  ability  of  bacteria  to 
form  indol.  Indol  is  formed  in  the  absence  of  utilizable  sugars  by 
Bacillus  coli;  members  of  the  cholera  group  and  other  bacteria  form 
tryptophan  by  the  splitting  off  of  alanin : 


CH2.CHNH2.COOH 


\     /\/ 

\/    NH 
Tryptophan 


Alanin.   The  alanin  is  decom- 
posed by  the  bacteria. 


1  New  York  Medical  Journal,  May  13,  1905. 

2  The  reaction  of  glycerin  broth  designed  for  the  cultivation  of  tubercle  bacilli  should 
be  +1.0  acid.    The  organism  does  not  develop  well  in  media  neutral  to  phenolphthalein. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      199 

Samples  of  Witte  peptone  occasionally  do  not  contain  tryptophan, 
consequently  each  lot  of  peptone  should  be  tested.  When  an  especially 
favorable  sample  is  found  it  should  be  reserved  for  this  purpose.  Plain 
neutral  sugar-free  broth  is  a  better  medium  than  Dunham's  solution 
for  the  indol  test,  and  it  should  be  employed  for  this  purpose  whenever 
possible. 

Nitrate  Broth. — Add  10  grams  of  Witte  peptone  to  one  liter  of  water 
and  dissolve  by  boiling.  Then  add  0.2  gram  chemically  pure  potas- 
sium nitrate — free  from  nitrites — and  filter.  Sterilize  in  the  autoclave. 
The  reaction  does  not  require  adjustment. 

Nutrient  Gelatin  Media. — Ten  grams  Witte  peptone  and  5  grams 
NaCl  are  added  to  1  liter  of  sugar-free  meat  infusion1  and  dissolved  by 
boiling.  When  the  ingredients  are  in  solution,  1002  grams  of  "Gold 
Label"  gelatin  are  added,  a  few  leaves  at  a  time,  and  stirred  until 
dissolved.  The  reaction  is  then  adjusted  to  the  desired  degree  and 
verified  after  an  additional  five  minutes'  heating.  The  medium  is 
cooled  to  50°  C.,  and  clarified  with  eggs,  using  two  eggs  for  each  liter. 
Filter  through  a  double  layer  of  absorbent  cotton  in  a  large  glass  funnel 
until  clear,  and  sterilize.  When  sterilization  is  accomplished,  cool 
quickly  and  store  in  the  ice-box. 

Nutrient  Agar. — (a)  Dissolve  12  grams  of  powdered  or  shredded  agar 
in  one  liter  of  meat  infusion  by  the  aid  of  heat  and  add  5  grams  NaCl 
and  10  grams  Witte  peptone.  Maintain  a  boiling  temperature  for  at 
least  thirty  minutes,  or  until  the  ingredients  are  completely  dissolved; 
restore  the  loss  by  evaporation,  adjust  the  reaction,  and  filter  through 
a  double  layer  of  absorbent  cotton  in  a  large  glass  funnel.  Pass  through 
filter  until  clear.  It  is  frequently  necessary  to  clarify  agar  with  eggs. 
After  the  reaction  is  adjusted,  cool  to  50°  C.  add  two  eggs  beaten  up 
in  water  and  mix  thoroughly.  Heat  slowly  to  the  boiling-point,  boil 
ten  minutes,  and  filter  through  absorbent  cotton;  sterilize  in  the 
autoclave. 

(b)  Prepare  " double-strength"  meat  infusion;  1000  grams  of  finely 
comminuted  lean  meat  are  suspended  in  one  liter  of  water;  infuse  in 
the  ice-box  for  twenty-four  hours;  heat  to  boiling  and  filter  through 
filter  paper.  Prepare  nutrient  meat  infusion  broth  with  this  strong 
infusion  as  a  basis  and  adjust  the  reaction  to  twice  the  desired  acidity— 
thus,  if  +1.0  is  to  be  the  final  reaction,  make  the  infusion  broth 

1  Meat  extract  may  be  used  in  place  of  meat  infusion,  but  the  medium  is  not  as  satis- 
factory for  pathogenic  bacteria. 

2  Use  120  grams  gelatin  during  warm  weather. 


200     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

+2.0.  Dissolve  24  grams  agar  in  one  liter  of  water,  boiling  steadily 
until  complete  solution  is  attained.  Add  to  the  meat  infusion  broth, 
boil  for  ten  minutes  and  clarify  with  eggs  in  the  usual  manner.  Filter 
and  sterilize. 

Meat  Extract  Agar. — Meat  extract  agar  is  made  by  substituting 
meat  extract  solution  for  meat  infusion;  otherwise  the  process  is  the 
same.  The  medium  must  be  clarified  with  eggs. 

Glycerin  Agar. — Five  per  cent,  of  glycerin  is  added  to  meat  infusion 
agar  immediately  before  filtration.  The  reaction  for  cultivation  of  the 
tubercle  bacillus  should  be  +1.0  acid  to  phenolphthalein.  Tubercle 
bacilli  do  not  thrive  in  media  neutral  to  phenolphthalein. 

Loffler's  Blood  Serum. — Add  one  part  by  volume  of  1  per  cent, 
nutrient  dextrose  broth1  to  three  parts  of  clear,  hemoglobin-free  beef 
or  sheep  serum,  and  distribute  in  test-tubes.  The  tubes  are  placed 
in  a  Koch's  serum  inspissator  or  in  specially  designed  racks  in  an  auto- 
clave in  an  inclined  position  to  produce  a  slanted  surface,  and  slowly 
heated  to  80°  C.  This  temperature  is  maintained  until  the  medium 
is  firmly  coagulated.  The  temperature  is  then  raised  to  95°  or  100° 
C.,  and  maintained  for  an  hour  on  each  of  three  successive  days,  or  to 
115°  in  the  autoclave,  and  maintained  for  one  hour.  The  medium  is 
opaque  and  white  and  the  surface  is  smooth  and  should  be  free  from 
a  metallic  lustre  when  viewed  by  reflected  light.  The  lustre  indicates 
an  accumulation  of  salts,  which  are  inimical  to  the  growth  of  many 
bacteria. 

Coagulated  Serum.2 — Clear  blood  serum  from  the  dog,  sheep,  cow.  or 
other  animal,  preferably  sterilized  by  filtration  through  Berkefeld 
filters,  and  with  or  without  the  addition  of  glycerin,  is  placed  in  test 
tubes  and  slanted  and  coagulated  in  a  serum  inspissator  at  a  tempera- 
ture of  75°  to  80°  C.  An  exposure  of  one  hour  to  this  temperature  on 
each  of  six  successive  days  is  necessary  to  insure  sterility.  The  medium 
should  be  translucent,  free  from  bubbles,  and  firm. 

Hiss  Serum  Water  Media. — Hiss3  has  recommended  a  serum  water 
medium  for  the  cultivation  of  pneumococci  and  similar  organisms. 
It  is  prepared  in  the  following  manner : 

Sheep  or  beef  serum,4  clear  and  free  from  hemoglobin,  is  added  to 
water  in  the  proportion  of  one  volume  of  serum  to  three  of  water. 

1  If  the  liquefaction  of  blood  serum  by  bacteria  is  to  be  tested,  sugar-free  broth  must 
be  used  in  place  of  dextrose-broth. 

2  Theobald  Smith,  Tr.  Am.  Phys.,  1898,  xiii,  417. 

3  Jour.  Exp.  Med.,  1905,  vii,  223. 

4  It  is  advisable  to  sterilize  the  serum  by  passage  through  an  unglazed  porcelain  filter. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      201 

Ten  per  cent,  aqueous  solutions  of  various  sugars  are  prepared  and 
sterilized,  and  a  sufficient  amount  of  the  desired  sugar  to  make  a 
final  concentration  of  1  per  cent,  is  added  to  the  sterile  serum  solution. 
Sufficient  sterile  5  per  cent,  litmus  solution  is  added  for  an  indicator. 
Fermentation  of  the  carbohydrate  is  shown  by  the  development  of  an 
acid  reaction,  and  frequently  by  a  well-defined  coagulation  of  the 
medium  as  well. 

Endo  Medium  for  the  Isolation  of  Typhoid,  Paratyphoid,  and  Dysentery 
Bacilli. — I.  Preparation  of  Agar. — (a)  Prepare  plain,  sugar-free  nutri- 
ent agar  as  described  on  page  197,  using  15  grams  of  agar  per  liter. 

(6)  Adjust  the  reaction  to  a  point  just  alkaline  to  litmus. 

(c)  Flask  the  agar,  100  c.c.  to  a  flask,  and  sterilize  in  the  autoclave. 

II.  Preparation  of  Indicator. — (a)  Prepare  a  10  per  cent,  solution 
of  basic  fuchsin  in  96  per  cent,  alcohol.    This  solution  is  fairly  stabile 
if  kept  away  from  the  light. 

(b)  Prepare  a  10  per  cent,  aqueous  solution  of  chemically  pure 
anhydrous  sodium  sulphite  (1  gram  in  10  c.c.  water).  This  solution 
does  not  keep. 

'  (c)  Add  1  c.c.  of  "II,  a"  to  10  c.c.  of  "II,  b"  and  heat  in  the  Arnold 
sterilizer  for  twenty  minutes.  The  color  of  the  fuchsin  is  nearly 
discharged  if  the  solutions  are  of  proper  strength.  This  solution  must 
be  prepared  each  day — it  does  not  keep. 

III.  Preparation  and    Use  of  Endomedium. — (a)  Add   1   gram  of 
C.  P.  lactose  (free  from  dextrose)  to  100  c.c.  of  agar  and  place  in  the 
autoclave  until  melted  and  the  lactose  is  thoroughly  dissolved. 

(b)  Add  a  sufficient  volume  of  "II,  c"  (about  1  c.c)  to  impart  a 
faint  pink  color  to  the  medium. 

(c)  Pour  into  sterile  Petri  dishes  and  allow  to  harden  in  a  dark 
place  with  the  covers  partly  removed.    When  cool  the  medium  should 
be  colorless  when  viewed  from  above  and  a  very  faint  pink  when  viewed 
from  the  edge.     The  medium  must  be  kept  in  a  dark  place  because 
the  color  is  restored  by  the  action  of  daylight. 

Those  bacteria  which  ferment  lactose — as  Bacillus  coli — form  lactic 
acid  which  restores  the  color  of  the  medium  in  the  immediate  neigh- 
borhood of  the  colony;  the  colony  therefore  is  colored  red.  Some 
aldehydes  also  restore  the  color,  but  it  is  not  very  probable  that  alde- 
hyde production  is  commonly  observed  among  the  lactose-fermenting 
organisms.  Non-lactose  fermenting  bacteria  grow  as  colorless  colonies. 

If  the  plates  are  to  be  incubated  two  or  three  days  it  may  be 
advisable  to  increase  the  agar  to  2.5  per  cent,  to  limit  the  diffusion  of 


202     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

color  from  the  acid  colonies.  For  rapid  isolations  the  medium  with 
the  normal  percentage  of  agar  is  preferable.1 

The  Technique  of  Inoculation  of  Endomedia  is  described  on  page  231. 

Lactose  Litmus  Agar. — I.  Prepare  1  per  cent,  lactose  nutrient  agar 
by  adding  10  grams  of  C.  P.  lactose  (free  from  dextrose)  to  one  liter 
of  plain  nutrient  agar.  Adjust  the  reaction  to  a  point  slightly  alkaline 
to  litmus.  Tube  and  sterilize  in  the  Arnold  sterilizer. 

II.  Prepare  an  aqueous  solution  of  litmus — either  a  5  per  cent, 
solution  of  purified  litmus  (Merck)  or  a  1  per  cent,  solution  of  azo- 
litmin  (Kahlbaum)  and  sterilize. 

To  Use  Lactose  Litmus  Agar. — Add  about  1  c.c.  of  sterile  litmus 
solution  to  a  sterile  Petri  dish  and  pour  over  it  the  melted  lactose 
agar,  previously  inoculated  with  the  desired  material.  For  water  and 
milk,  add  1  c.c.  of  water  or  milk  (diluted  to  the  proper  degree)  to  the 
Petri  dish  before  adding  the  lactose  agar.  Mix  intimately  by  rotating 
gently,  allow  to  harden,  and  incubate. 

Those  bacteria  which  ferment  lactose  with  the  production  of  acid 
appear  as  red  colonies.  Non-lactose-fermenting  organisms  appear  as 
blue  colonies. 

Blood  Agar. — Blood  is  drawn  with  aseptic  precautions  from  the 
carotid  or  femoral  artery  of  a  dog  or  rabbit  into  a  sterile  flask  con- 
taining beads.  The  blood  is  defibrinated  by  prolonged  agitation  and 
added  to  plain  (not  dextrose)  nutrient  agar  previously  melted  and 
cooled  to  45°  C.,  in  the  proportion  of  2  c.c.  of  blood  to  10  c.c.  of  agar. 
Small  amounts  of  blood  may  be  withdrawn  directly  from  the  heart 
of  an  animal  without  difficulty,  provided  a  small  hypodermic  needle 
is  used.  The  blood  may  be  injected  directly  into  the  melted  agar 
without  defibrination. 

Occasionally  human  blood  is  added  to  agar;  if  a  series  of  agar  slants 
are  prepared  it  is  possible  to  convert  them  into  blood  agar  with  a  small 
amount  of  blood,  as  follows : 

Withdraw  10  c.c.  of  blood,  using  aseptic  -precautions,  from  the 
median  basilic  vein,  in  a  large  syringe.  Inject  the  blood  at  once  into 
four  times  the  volume  of  plain  nutrient  agar  melted  and  cooled  to  45° 
C.  Mix  at  once  and  run  2  c.c.  over  the  slanted  surface  of  each  agar 
slant,  and  allow  to  harden  in  the  inclined  position  in  such  a  manner 
that  a  uniform  layer  of  the  blood-agar  mixture  is  obtained.  Incubate 
to  prove  sterility. 

1  Kendall  and  Day,  Jour.  Med.  Res.,  1911,  xxv,  95. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      203 

Ascitic  and  Hydrocele  Fluid  Media. — Ascitic  and  Hydrocele  Agar.1— 
Collect  ascitic  or  hydrocele  fluid  in  a  sterile  bottle,  using  aseptic 
precautions.  Allow  to  stand  in  the  ice-box  until  clear,  and  heat  to 
50°  C.  for  half  an  hour  to  destroy  enzymes.  Two  parts  of  hydrocele 
or  ascitic  fluid  to  eight  or  ten  parts  of  plain  nutrient  agar  previously 
melted  and  cooled  to  45°  C.  make  a  medium  especially  adapted  to  the 
growth  of  many  of  the  more  fastidious  pathogenic  bacteria.2 

Ascitic  broth  is  prepared  by  adding  20  to  50  per  cent,  by  volume  of 
sterile  ascitic  fluid  to  plain  nutrient  broth.  Incubate  to  prove  sterility. 

Egg  Media. — Eggs  are  a  very  good  substitute  for  blood  serum  in 
Loffler's  medium.  Eggs  are  carefully  broken  into  a  clean  beaker 
stirred  gently  with  a  rod  (avoiding  the  formation  of  air  bubbles)  until 
homogeneous,  and  mixed  with  dextrose  broth  in  the  proportion  of 
one  part  by  volume  of  broth  to  three  volumes  of  egg.  The  medium 
is  coagulated  in  a  slanted  position  and  sterilized  precisely  as  Loffler's 
blood  serum  is  coagulated  and  sterilized. 

Egg  Medium. — No.  1.  Mix  four  to  six  volumes  of  thoroughly  homo- 
genized eggs  with  one  volume  of  nutrient  broth,  and  add  sufficient 
glycerin  to  make  the  concentration  of  the  latter  3  per  cent,  by  weight. 
Coagulate  and  sterilize  in  the  slanted  position  precisely  as  Loffler's 
blood  serum  is  coagulated  and  sterilized.  This  medium  is  excellent 
for  the  cultivation  of  tubercle  bacilli. 

No.  2.  Add  one  volume  of  physiological  salt  solution  to  ten  volumes 
of  egg  which  have  been  lightly  stirred  with  a  rod  until  the  yolks  and 
whites  are  intimately  incorporated.  Coagulate  and  sterilize  in  a 
slanted  position  in  test  tubes. 

Milk  and  Litmus  Milk. — One  liter  of  fresh  milk  is  thoroughly  mixed 
and  tubed  in  the  ordinary  manner.  Litmus  milk  is  prepared  by  adding 
sufficient  litmus  solution  to  impart  a  clea'r  blue  color.  It  is  tubed, 
using  10  c.c.  to  each  tube,  and  sterilized  in  the  autoclave. 

For  some  purposes  it  is  desirable  to  remove  the  cream  before  tubing, 
but  for  cultural  work  the  color  of  the  cream  ring  in  litmus  milk  is  of 
some  diagnostic  importance.  Thus,  members  of  the  paratyphoid 
group  of  bacilli  almost  invariably  show  a  blue-green  cream  ring;  the 
colon  bacillus  colors  the  cream  ring  red  brown.  It  should  be  remem- 
bered that  litmus  milk  does  not  coagulate  as  readily  or  as  rapidly  as 
plain  milk. 

1  Ascitic  and  hydrocele  fluids  may  be  sterilized  by  passage  through  an  unglazed  porce- 
lain filter. 

2  It  should  be  remembered  that  ascitic  and  hydrocele  fluids  usually  contain  about 
0.08  per  cent,  dextrose. 


204     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

Potato. — New  potatoes  have  an  acid  reaction,  as  a  rule,  and  old 
potatoes  are  slightly  alkaline. 

Large  potatoes  are  thoroughly  scrubbed,  the  skin  removed,  and 
cut  into  cylinders  with  a  cork-borer.  The  cylinders,  which  should  be 
at  least  1.5  cm.  in  diameter,  are  divided  into  equal  parts  by  a  diagonal 
cut.  The  pieces  are  placed  in  running  water  overnight  so  that  they 
will  not  darken,  and  are  inserted,  base  downward,  in  large  test  tubes. 
It  is  advisable  to  add  about  1  c.c.  of  water  to  each  tube  to  prevent 
drying.  Sterilize  in  the  autoclave. 

Hiss's1  Semisolid  Medium.— 

FORMULAE 

Water    .      .     -. 1000  c.c. 

Agar 8  grams 

Peptone 10      " 

Meat  extract 3      " 

NaCl 5      " 

Gelatin2 40      " 

When  all  the  ingredients  are  dissolved,  adjust  the  reaction  to  +0.5 
(phenolphthalein),  filter,  and  add  sufficient  litmus  solution  to  impart 
a  clear  blue.  Dissolve  1  per  cent,  of  dextrose,  lactose,  saccharose, 
mannite,  or  other  carbohydrate  in  the  medium,  and  fill  test-tubes 
with  it.  Sterilization  of  lactose  and  saccharose  semisolid  media  is 
preferably  carried  out  in  the  Arnold  sterilizer.  Dextrose  and  mannite 
media  may  be  sterilized  in  the  autoclave. 

Semisolid  media  are  inoculated  by  the  stab  method.  A  change  in 
reaction  is  indicated  by  the  litmus;  gas-forming  organisms  form  bubbles 
in  the  depth  of  the  medium. 

Russell  Double  Sugar  Medium. — To  1  liter  of  nutrient  agar,  slightly 
alkaline  to  litmus,  add  sufficient  sterile  5  per  cent,  litmus  solution  to 
impart  a  distinct  clear  blue  color.  Add  1  per  cent,  of  C.  P.  lactose 
and  0.1  per  cent,  dextrose,  and  distribute  in  test-tubes. 

Sterilize  in  the  Arnold  sterilizer  for  three  successive  days,  and  allow 
to  harden  in  a  slanted  position. 

Media  for  the  Cultivation  of  Aciduric  Bacteria. — Acid  Broth. — Add 
sufficient  glacial  acetic  acid  to  a  liter  of  2  per  cent,  dextrose  broth 
to  make  the  reaction  equal  to  50  c.c.  of  normal  acid.  A  precipitate 
forms,  which  will  settle  out,  leaving  a  clear  supernatant  fluid  that  may 
be  removed  to  sterile  test  tubes  with  a  sterile  10  c.c.  pipette. 

Oleate  Agar. — The  addition  of  0.2  per  cent,  sodium  oleate  to  dextrose 
agar  makes  a  favorable  medium  for  the  cultivation  of  aciduric  bacteria. 

1  Jour.  Exp.  Med.,  1897,  ii,  677. 

2  Add  after  the  other  ingredients  are  in  solution. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      205 

V.  The  Cultivation  of  Bacteria. — Inoculation  of  Culture  Media.— 
A  platinum  wire  of  24-gauge  is  generally  used  to  transfer  bacteria  from 
medium  to  medium.  A  piece  of  platinum  wire1  three  inches  in  length 
is  fused  into  the  end  of  a  glass  rod  5  mm.  in  diameter  and  about  15 
cm.  in  length.  Metal  handles  are  preferred  by  many;  they  possess 
the  great  advantage  of  not  breaking,  but  become  heated  during  the 
process  of  sterilization.  The  straight  wire  or  "  needle"  is  commonly 
used  for  the  inoculation  of  slant  and  stab  cultures  in  solid  media; 
for  the  inoculation  of  fluid  media  a  loop  is  formed  on  the  end  of  the 
wire.  The  use  of  the  loop  permits  of  the  transfer  of  a  greater  amount 


FIG.  23.— Needle  sterilizer.     (A.  de  Khotinsky.) 

of  material.  It  is  occasionally  necessary  to  transfer  more  material 
than  a  drop  or  two  obtained  with  a  loop  in  order  to  insure  growth, 
and  for  this  purpose  sterile  capillary  pipettes  are  very  convenient. 
Many  anerobic  bacteria  and  organisms  which  grow  poorly  in  artificial 
media  must  be  transferred  with  the  pipette. 

The  transfer  of  bacteria  from  media  to  media  involves  the  following 
steps : 

(a)  Flame  cotton  plugs  to  destroy  molds  and  spores  of  bacteria; 
extinguish  flame. 

(6)  Twist  cotton  plugs  to  ^destroy  adhesion  to  the  neck  of  the 
tube.  The  plugs  may  then  be  removed  intact. 

(c)  Sterilize  platinum  wire  in  Bunsen  flame.     Heat  wire  white  hot 
and  pass  that  portion  of  the  handle  adjoining  the  wire  through  the 
flame,  rotating  it  between  the  fingers  while  doing  so.    Allow  the  wire 
to  cool. 

(d)  Grasp  the  tubes  in  the  left  hand  and  remove  plugs  from  the 
tubes,  holding  one  between  the  third  and  fourth  fingers  of  the  right 

1  A  cheap  and  efficient  substitute  for  platinum  wire  is  "Nichrome"  wire.  It  is  rather 
less  durable  than  platinum,  and  melts  at  a  lower  temperature. 


206     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 


hand,  the  other  between  the  second  and  third  fingers,  the  plugs  pro- 
jecting outward.  Flame  the  mouths  of  the  tubes  and  test  coolness 
of  the  platinum  wire  by  plunging  it  for  a  distance  of  about  a  centi- 
meter into  the  sterile  medium. 

(e)  Remove  some  material  from  the  infected  tube  by  dipping  the 
tip  of  the  wire  in  it,  and  transfer  to  the  sterile  tube. 

(/)  Replace  the  plugs  in  their  respective 
tubes  and  sterilize  the  wire  before  laying  it 
down. 

II.  The  Isolation  of  Pure  Cultures  of  Bacteria. 
— A.  Aerobic  Organisms. — It  is  the  exception 
rather  than  the  rule  that  bacteria  exist  in  nature 
or  in  many  pathological  processes  in  pure  cul- 
ture, that  is,  that  a  single  kind  of  organism 
alone  is  present.  From  such  mixtures  of  bac- 
teria it  is  frequently  necessary  to  isolate  one  or 
more  organisms  in  a  pure  state,  uncontami- 
nated  by  other  microorganisms.  A  common 
and  efficient  method  of  separating  bacteria  from 
mixtures  is  to  distribute  them  in  melted  gelatin 
or  agar,1  in  such  a  manner  that  individual  cells 
are  somewhat  widely  separated.  The  medium 
is  then  allowed  to  harden.  The  organisms  are 
immobilized  in  or  upon  the  medium  and  sur- 
rounded by  nutrients;  the  descendants  of  each 
individual  organism  thus  develop  locally  and 

apart  from  the  descendants  of  other  organisms.  Under  favorable 
conditions  the  descendants  of  individual  cells  become  so  numerous 
they  may  be  seen  with  the  unarmed  eye  as  spots  or  colonies,  each 
of  which  is  made  up  of  the  progeny  of  a  single  organism.  It  is  a 
simple  matter  to  touch  such  a  colony  with  a  sterile,  cool  platinum 
needle,  and  infect  sterile  media  with  the  adherent  bacteria.  In  this 
manner  pure  cultures  are  obtained.  The  technic  of  the  isolation  of 
aerobic  and  facultatively  anaerobic  bacteria  is  technically  termed 
plating,  or  streaking,  depending  upon  the  apparatus  used. 

1.  Plate  Method. — Three  tubes  of  nutrient  agar  or  gelatin  are  melted 
and  cooled  to  42°  to  45°  C.  A  platinum  wire,  previously  sterilized 

1  Agar  melts  at  about  95°  C.  and  solidifies  at  about  40°  C.  It  is  necessary  to  work 
rapidly  with  melted  and  cooled  agar,  to  carry  out  the  technic  of  inoculation  before 
solidification  takes  place. 


FIG.  24. — Platinum  needle 
and  platinum  loop. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      207 

and  cooled,  is  dipped  to  a  depth  of  about  0.5  c.c.  in  a  mixture  of  bac- 
teria in  fluid  media,  or  touched  to  a  growth  in  solid  media,  and  then 
rotated  two  or  three  times  in  a  tube  of  the  sterile  melted  medium. 
Without  sterilizing  the  needle,  the  process  is  repeated  in  the  second 
and  third  tubes.  Each  tube  is  then  rotated  between  the  palms  of  the 
hands,  to  distribute  the  organisms  thoroughly,  and  poured  individually 
into  the  sterile  Petri  dishes.  The  medium  is  flowed  uniformly  over 
the  bottom  of  the  dish  and  set  aside  to  harden. 

It  will  be  seen  that  the  first  tube  inoculated  contains  the  greatest 
number  of  organisms,  and  that  the  third  tube  would  theoretically 
contain  but  few.  The  colonies  in  one  of  the  plates  will  be  so  widely 
separated  that  they  can  be  "fished"  with  the  platinum  wire  without 
the  danger  of  touching  other  colonies,  and  transferred  to  fresh,  sterile 
media.  The  success  of  this  procedure  depends  largely  upon  a  rigorous 
observance  of  details.  The  mouths  of  the  tubes  and  the  cotton  plugs 
should  be  flamed  thoroughly  before  inoculation  is  practiced,  and  the 
transfer  of  the  contents  of  the  tube  to  the  Petri  dish  must  be  done 
carefully  to  prevent  contamination.  *The  cover  of  the  Petri  dish  should 
be  raised  with  the  left  hand,  but  directly  over  the  bottom,  to  prevent 
the  entrance  of  adventitious  bacteria  from  the  air.  The  mouth  of  the 
tube  should  not  touch  the  bottom  or  edge  of  the  Petri  dish  and,  finally, 
the  cover  of  the  latter  should  be  replaced  at  once. 

After  the  medium  has  hardened  the  plates  are  incubated — gelatin 
plates  at  20°  C.,  agar  plates  at  37°  C.  It  is  customary  to  invert  agar 
plates  during  incubation;  when  agar  cools  and  becomes  solid  a  con- 
traction takes  place  which  squeezes  out  some  fluid.  (This  is  well 
defined  in  slanted  agar  as  the  water  of  condensation.).  If  the  fluid 
were  allowed  to  remain  on  the  surface  of  the  agar  plate  it  would  con- 
vert the  surface  potentially  into  a  broth  culture,  in  which  the  various 
organisms  would  mix  in  hopeless  confusion.  Inversion  of  the  plates 
prevents  the  accumulation  of  moisture  on  the  surface  to  a  large  degree; 
the  water  of  condensation  collects  on  the  cover  instead.  The  porous 
tops  recommended  by  Hill  may  advantageously  be  used — they  absorb 
moisture  as  it  is  formed.  Gelatin  plates  are  not  inverted;  fluid  is 
not  expressed  as  the  medium  solidifies,  and  liquefied  gelatin  formed 
during  the  growth  of  actively  proteolytic  organisms  would  collect  on 
the  cover  and  probably  contaminate  the  entire  plate. 

2.  Streak  Method. — The  isolation  of  pure  cultures  of  bacteria  by  the 
streak  method  differs  from  the  plate  method  in  that  the  medium 
(gelatin,  agar,  blood  serum)  is  not  inoculated  in  the  fluid  state;  the 


208     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

necessary  dilution  to  secure  isolated  colonies  is  attained  by  drawing 
a  platinum  needle  infected  with  bacteria  several  times  across  the  sur- 
face of  sterile,  slanted  gelatin,  agar,  blood  serum,  blood  agar  or^  other 
solid  medium,  each  time  covering  an  area  not  previously  touched. 
Eventually  a  degree  of  dilution  is  reached  where  discrete  colonies  are 
discernible. 

The  plating  method  and  streak  method  possess  advantages  and 
disadvantages.  A  considerable  proportion  of  the  growth  in  plates 
inoculated  in  the  fluid  state  is  beneath  the  surface,  where  it 
is  less  characteristic  than  surface  colonies.  The  distribution  of 
organisms,  however,  is  more  uniform,  and  small  numbers  of  bacteria 
occurring  in  mixture  with  larger  numbers  of  undesirable  organisms 
are  somewhat  less  likely  to  be  overlooked.  It  is  possible,  moreover, 
to  obtain  a  quantitative  estimation  of  the  numbers  of  bacteria  in 
mixtures  by  the  plate  method.  The  streak  method  is  advantageous 
both  with  respect  to  the  economy  of  time  necessary  to  inoculate  the 
medium,  and  in  that  the  colonies  are  wholly  upon  the  surface  of  the 
medium.  There  is  less  danger  of  contamination  when  u  fishing"  from 
streak  plates  than  from  the  regular  method  of  plating,  because  there 
is  no  chance  for  submerged  colonies  to  underlie  those  upon  the  surface. 

The  use  of  certain  kinds  of  media,  as  that  of  Endo,  of  blood  agar, 
and  Loffler's  blood  serum,  requires  that  surface  inoculation  shall  be 
made.  The  possibility  of  missing  or  overlooking  small  numbers  of 
the  less  hardy  types  of  bacteria  is  greater  with  the  streak  method  of 
isolation. 

3.  The  Barber  Method  for  the  Isolation  of  a  Single  Cell. — It  is  occa- 
sionally necessary,  in  very  refined  bacteriological  studies,  to  be  abso- 
lutely certain  that  the  starting  point  of  a  pure  culture  is  a  single 
organism.  Theoretically,  single  cells  are  the  progenitors  of  the  colonies 
observed  in  media  inoculated  by  the  plate  or  the  streak  method,  and 
such  is  usually  the  case.  Undoubtedly  it  may  happen  that  a  chain  of 
streptococci  may  remain  adherent  and  their  descendants  appear  as  a 
single  colony,  and  it  is  equally  certain  that  two  alien  bacteria  may 
occasionally  become  adherent  by  intertwining  of  flagella  or  adhesion 
of  viscid  capsular  substance  and  develop  into  a  mixed  colony.  The 
apparatus  of  Barber,1  which  consists  essentially  of  a  delicate  capillary 
pipette  mounted  in  the  substage  of  the  microscope,  and  capable 
of  upward  and  downward  motion  in  the  optical  axis  of  the  instrument, 
is  designed  to  circumvent  this  possibility.  In  practice  a  very  thin 

1  Univ.  Kansas  Science  Bull.,  No.  1,  March,  1907. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      209 

emulsion  of  bacteria  in  a  fluid  medium  is  placed  on  the  surface  of  a 
sterile  thin  plate  of  glass  in  such  a  manner  that  a  drop  of  the  con- 
taminated fluid  hangs  in  the  opening  in  the  stage  ordinarily  occupied 
by  the  condenser.  The  drop  is  manipulated  by  a  mechanical  stage, 
guided  by  direct  observation  with  a  one-sixth-inch  lens  until  a  single 
organism  appears  in  the  field  of  vision.  The  sterile  capillary  pipette 
is  carefully  brought  upward  until  the  tip  engages  the  dependent  drop; 
the  organism  will  be  seen  to  enter  the  pipette,  which  is  then  lowered  and 
removed  from  its  attachments  to  the  microscope.  The  single  cell  is 
transferred  to  a  suitable  medium  and  incubated  in  the  usual  manner. 

B.  Anaerobic  Bacteria. — 1.  Plating  Methods. — The  cultivation  of 
anaerobic  bacteria  which  do  not  grow  in  the  presence  of  atmospheric 
(free)  oxygen  requires  special  apparatus  and  technic.  The  simplest 
method,  and  one  which  is  successful  if  gas-forming  bacteria  are  absent; 
is  to  make  dilutions  in  dextrose  agar  precisely  as  described  under 
Plating  in  the  preceding  paragraphs.  The  tubes  should  be  filled  to  a 
depth  of  10  cm.  with  the  medium,  and  tubes  of  relatively  large  dia- 
meter— 2  to  3  cm. — are  preferable.  The  tubes,  previously  heated  to 
the  boiling  point,  and  rapidly  cooled  to  43  to  45°  C.  to  prevent  reabsorp- 
tion  of  oxygen,  are  inoculated  by  the  dilution  method,  rotated  between 
the  hands  to  distribute  the  organisms  uniformly,  and  cooled  rapidly 
in  an  upright  position. 

Colonies  appear  within  the  depths  of  the  media  after  incubation; 
in  the  thinly  seeded  tubes  these  colonies  are  discrete,  and  they  may 
be  removed  without  contamination,  either  in  sterile  capillary  pipettes 
introduced  through  the  surface  of  the  medium,  or  after  breaking  the 
tubes  from  the  side.  It  is,  of  course,  necessary  to  sterilize  the  outside 
of  the  tube  if  it  is  to  be  broken.  A  greater  degree  of  anaerobiosis  may 
be  obtained  within  the  tubes  if  after  solidifying  they  are  placed  neck 
downward  with  the  cotton  plugs  removed,  in  a  beaker  containing 
freshly  prepared  alkaline  pyrogallate  solution.1  Growth  of  anaerobic 
bacteria  upon  the  surface  of  agar  or  blood  serum  may  be  obtained  in 
this  manner.2  Those  bacteria  which  produce  gas  during  their  growth 
cannot  be  isolated  in  pure  culture  in  deep  agar  tubes;  the  liberation 
of  gas  bubbles  fragments  the  medium  and  permits  the  various  colonies 
to  coalesce. 

1  Five  grams  of  dry  pyrogallic  acid  are  placed  in  a  beaker  and  covered  with  15-25  c.c. 
of  water:  when  dissolved  a  layer  of  kerosene  or  paraffin  oil  about  1  cm.  in  depth  is  added, 
and  a  10  per  cent,  solution  of   sodium  hydroxide  is  introduced  below  the  oil  layer  with 
a  pipette. 

2  See  Rickards,  Cent.  f.  Bakt.,  1904,  I  Abt.,  xxxvi,  557. 

14 


210     MICROSCOPIC   AND  CULTURAL  STUDY  OF  BACTERIA 


The  "  bottle-plate"  method  of  Simonds  and  Kendall1  overcomes  this 
difficulty  to  a  considerable  degree — through  the  use  of  simple  appli- 
ances. 

Sixteen-ounce  French  square  tincture-mouth  bottles  are  plugged 
with  cotton  and  sterilized  with  dry  heat.  With  the  bottles  lying  on 
their  sides,  sufficient  blood  agar  is  poured  in  to  form  a  layer  5  to  10 

mm.  deep,  and  allowed  to  harden.  Dorset's 
egg  medium,  dextrose  agar  or  other  media 
may  be  substituted  for  the  blood  agar  if 
desired. 

As  soon  as  the  medium  has  hardened  the 
bottles  should  be  turned  on  the  opposite 
side,  thus  bringing  the  medium  uppermost 
and  preventing  condensation  water  from 
adhering  to  it.  Inoculation  is  made  with  a 
bent  glass  rod  infected  with  bacteria  from 
a  thin  suspension  in  a  liquid  medium,  and 
rubbed  over  the  surface  of  the  agar  within 
the  bottle.  A  partial  vacuum  is  next  cre- 
ated within  the  bottle,  and  residual  oxygen 
dissolved  in  alkaline  pyrogallate  solution 
in  the  following  manner:  A  closely  fitting 
rubber  stopper  with  one  hole  carrying  a 
glass  tube  four  inches  in  length  is  inserted 
in  the  bottle.  The  outer  end  of  the  glass 
tube  projects  three-quarters  of  an  inch 
beyond  the  stopper  and  is  fitted  with  a 
rubber  tube  three  inches  in  length.  That 
portion  of  the  glass  rod  within  the  bottle 
is  bent  at  an  angle  of  45°  and  the  stopper 
is  turned  in  such  a  manner  that  the  end  of 
the  glass  tube  points  toward  the  side  of  the 
bottle  opposite  the  layer  of  agar.  As  much 

air  as  possible  is  aspirated  from  the  bottle,  and    the  rubber  tube 
closed  with  a  pinch-cock  to  prevent  reentrance  of  air. 

The  bottle  is  now  placed  on  its  side,  with  the  medium  uppermost, 
and  with  a  pipette,  10  c.c.  each  of  a  50  per  cent,  solution  of  pyrogallic 
acid  and  10  per  cent,  sodium  hydrate  are  run  in  through  the  rubber 
tube,  avoiding  the  entrance  of  air.  A  few  cubic  centimeters  of  clean 

1  Jour.  Inf.  Dis.,  1912,  xi,  207. 


FIG.  25. — Wright's  method  of 
making  anaerobic  cultures  in 
fluid  media.  (Mallory  and 
Wright.) 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      211 

water  are  also  run  in,  to  wash  the  rubber  tube  free  from  caustic  alkali. 
The  apparatus  is  working  properly  when  the  rubber  tube  between  the 
pinch-cock  and  the  bottle  is  collapsed,  indicating  a  partial  vacuum 
within  the  bottle  itself.  Residual  oxygen  is  rapidly  absorbed  within 
the  pyrogallate  solution,  leaving  an  inert  atmosphere  of  nitrogen. 

The  bottle  is  incubated,  medium  uppermost,  for  the  required  time. 
Inspection  of  the  surface  of  the  medium  will  show  the  colonies.  After 
incubation  the  pinch-cock  is  carefully  opened,  admitting  air  very  gently 
to  avoid  spattering  the  medium,  and  the  stopper  is  removed.  The 
pyrogallate  solution  is  poured  out  and  residual  traces  removed  with 
clean  water.  The  bottle  is  drained  standing  upon  end,  mouth  down, 
and  then  the  colonies  are  ready  for  fishing.  The  colonies  which  develop 
are  all  surface  growths :  the  isolation  of  gas-forming  anaerobic  bacteria 
is  as  readily  accomplished  as  the  isolation  of  non-aerogenic  types. 


FIG.  26. — Novy  jar  for  anaerobic  cultures.     (Park.) 

Pure  cultures  of  anaerobic  bacteria  may  be  obtained  in  an  atmos- 
phere of  hydrogen;  plates  prepared  in  the  usual  manner  are  placed 
on  a  rack  in  a  Novy  jar  or  other  similar  vessel  provided  with  a  tightly 
fitting  stop-cock,  through  which  hydrogen  can  be  admitted  in  sufficient 
volume  to  displace  the  air.  The  stop-cock  must  be  hydrogen-tight. 
The  procedure  is  to  place  inoculated  plates  without  covers  on  a  rack 
within  the  jar  in  an  inverted  position,  one  above  the  other.  A  few 
grams  of  pyrogallic  acid  are  placed  on  the  bottom  of  the  jar  with  a 
small  piece  of  solid  sodium  hydroxide.  At  the  last  moment,  when 
everything  is  in  readiness,  20  to  30  c.c.  of  water  are  gently  poured 
down  the  side  of  the  jar  to  prevent  spattering,  and  the  cover  quickly 
clamped  down.  A  current  of  hydrogen  gas,  either  from  a  cylinder  or 
from  a  Kipp  generator,  is  passed  through  the  jar  at  a  fairly  rapid  rate. 


212     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

The  hydrogen  should  enter  at  the  top,  and  the  outlet  for  the  gas  should 
be  as  near  the  bottom  of  the  apparatus  as  possible.  A  sample  of  the 
escaping  gas,  collected  in  a  test-tube  by  downward  displacement, 
will  ignite  without  an  explosion  when  all  oxygen  is  displaced.  The 
inlet  tubes  are  closed,  and  incubation  practiced  in  the  usual  manner. 
An  atmosphere  of  nitrogen  is  to  be  preferred  to  an  atmosphere  of 
hydrogen  whenever  it  is  practicable. 

b          c®  d 

af 


FIG.  27.— Koch  i&fety 
burner.     (Park.) 


FIG.  28. — Dunham  thermo- 
regulator.     (Park.) 


FIG.  29. — Roux   Bimetallic 
regulator.     (Park.) 


2.  Anaerobic  Cultures  in  Fluid  Media. — A  simple  method  of  main- 
taining anerobiosis  in  fluid  media,  sufficiently  effective  for  ordinary 
usage,  is  to  overlay  a  flask  or  test  tube  containing  dextrose  broth 
with  a  layer  of  albolene  about  1  cm.  in  depth.  Immediately  before 
inoculation  all  residual  oxygen  in  the  medium  should  be  removed 
by  an  exposure  of  half  an  hour  in  the  Arnold  sterilizer,  or  ten  minutes 
in  an  autoclave.  The  liquid  is  cooled  rapidly  to  minimize  reabsorp- 
tion  of  oxygen.  Wright1  has  maintained  anaerobic  conditions  in  test- 
tube  cultures  with  alkaline  pyrogallate  solution.  Test  tubes  are 
prepared  with  absorbent  cotton  plugs,  which  are  made  tighter  than 
ordinary  usage  demands.  After  the  culture  medium  (freed  from  dis- 
solved oxygen  by  heating  and  rapid  cooling)  is  inoculated,  the  cotton 


1  Mallory  and  Wright,  Pathological  Technic,  4th  ed.,  p.  126. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      213 

plug  is  pushed  into  the  tube  until  the  upper  end  is  about  15  mm. 
below  the  top.  The  space  above  the  cotton  plug  is  filled  loosely  with 
dry  pyrogallic  acid  and  a  strong  solution  of  sodium  hydroxide,  2  to 
3  c.c.,  is  added  to  dissolve  the  acid.  Immediately  a  tightly  fitting 
rubber  stopper  is  inserted  into  the  mouth  of  the  tube.  The  alkaline 
pyrogallate  solution  absorbs  the  oxygen  within  the  tube,  leaving  an 
atmosphere  of  nitrogen. 


FIG.  30.— Incubator.     (Park.) 

The  addition  of  bits  of  fresh,  sterile  tissue,1  fresh,  sterile  defibrinated 
blood,  or  of  the  coagulum  which  is  formed  during  the  coagulation  of 
meat  infusion  adds  greatly  to  the  nutritional  value  of  cultures  for  the 
growth  of  anaerobic  bacteria. 

1  Theobald  Smith,  Cent.  f.  Bakt.,  1890,  vii,  502. 


214     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

Special  mention  of  the  preparation  of  tissue  media  is  made  in  the 
sections  on  Specific  Anaerobic  Organisms. 

The  Incubation  of  Bacterial  Cultures. — The  growth  of  bacteria  in 
artificial  media  is  markedly  influenced  by  the  temperature  to  which 
they  are  exposed.  A  majority  of  those  organisms  parasitic  upon  or 
pathogenic  for  man  develop  most  luxuriantly  at  the  temperature  of 
the  human  body,  37°  C.  Exposure  to  temperatures  but  slightly  above 
37°  C.  leads  to  rapid  death  of  these  organisms,  consequently  incuba- 
tors must  be  available  within  which  cultures  may  be  safely  exposed 
to  a  uniform  and  constant  degree  of  heat  equal  to  that  of  the  human 
body.  Gelatin  cultures  must  be  maintained  at  temperatures  not 
exceeding  22°  C. 

Incubators  are  single-  or  double-walled  chambers  of  various  sizes, 
heated  directly  by  gas  or  electricity,  or  indirectly  through  a  water 
jacket.  The  latter  run  more  uniformly,  because  water  receives  and 
imparts  heat  more  slowly  than  air.  On  the  other  hand,  large  incuba- 
tors cannot  be  surrounded  with  water  jackets  because  of  mechanical 
difficulties.  The  regulation  of  temperature  within  incubators  is  con- 
trolled by  bimetallic  regulators  which  actuate  valves  or  electromagnets 
controlling  the  supply  of  gas  or  electricity  which  heats  the  chamber, 
or  by  mercurial  thermoregulators  working  upon  the  principle  of  the 
mercury  thermometer.  Bimetallic  regulators,  in  which  the  movement 
imparted  to  the  regulator  of  the  source  of  supply  of  heat  is  due  to  the 
differential  expansion  or  contraction  of  two  dissimilar  metals,  are  more 
sensitive  to  slight  variations  in  heat  and  they  possess  the  additional 
advantage  of  being  less  fragile  than  mercurial  regulators.  Various 
patterns  of  thermoregulators  of  tried  efficiency  are  on  the  market  and 
a  selection  between  them  is  largely  a  matter  of  mechanical  adaptability 
to  local  needs. 

VI.  The  Study  of  Bacterial  Cultures. — I.  Growth  in  Solid  Media.— 
(a)  Colonies. — The  macroscopic  appearance  of  bacterial  colonies  upon 
solid  media  is  of  considerable  value  for  the  differentiation  and  recog- 
nition of  the  various  types;  in  a  similar  manner  their  microscopic 
appearance,  stained  or  unstained,  permits  of  some  differentiation. 

The  aspect  of  a  colony  is  influenced. 

1.  By  the  kind  of  organism — Streptococcus  colonies,  for  example, 
are  habitually  small  and  nearly  transparent;  anthrax  colonies  are 
habitually  larger  and  opaque. 

2.  By  the  consistency  of  the  medium — in  firm,  dense  media  the 
growth  of  bacteria  is  limited  and  relatively  dry;  in  moist,  semisolid 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      215 

media  the  growth  of  the -same  organism  is  usually  luxuriant,  moist, 
and  spreading. 

3.  By  the  composition  and  reaction  of  the  medium — the  addition 
of  specifically  nutritive  substances,  as  of  fresh  sterile  tissue  to  media 
for  the  cultivation  of  anaerobes;  of  utilizable  carbohydrates  to  media 
for  the  cultivation  of  carbohydrophilic  bacteria;  of  fresh,  defibrinated 
blood  to  media  for  the  cultivation  of  hemoglobinophilic  organisms; 
these  may  improve   conditions  otherwise   unfavorable   for  bacterial 
development. 

The  reaction  of  the  medium,  furthermore,  is  important;  many 
bacteria  are  extremely  sensitive  to  slightly  acid  media;  the  aciduric 
bacteria  thrive  in  media  too  acid  for  the  existence  of  other  organisms. 
Even  the  ordinary  laboratory  media,  made  according  to  a  definite 
formula,  vary  sufficiently  in  chemical  and  physical  properties  to 
influence  materially  the  appearance  of  bacterial  colonies.  The  degree 
of  influence  is  more  pronounced  in  the  feebly  growing  forms,  but  it 
may  affect  the  appearance  of  colonies  of  the  more  hardy  types  as  well. 

4.  The  rate  of  growth  of  bacteria  also  affects  the  appearance  of 
colonies. 

It  is  useless,  as  a  scientific  procedure,  to  attempt  to  recognize  dif- 
ferences of  greater  refinement  than  the  accuracy  of  the  method  permits 
of,  and  for  this  reason  the  descriptions  of  bacterial  colonies  should 
not  be  carried  to  extremes.  In  general,  bacterial  growths  on  solid 
media  are  described  as  solids  in  space — the  average  size,  form,  color, 
lustre  and  texture.  This  applies  equally  well  to  colonies,  slant  and 
stab  cultures.  The  really  valuable  information  gleaned  from  a  study 
of  bacterial  growths  is  the  recognition  of  types  of  growth.  For  example, 
spore-forming  bacteria  (aerobic)  produce  rather  heavy,  opaque,  floc- 
culent  colonies;  members  of  the  Alcaligenes — dysentery,  typhoid, 
paratyphoid  group — grow  characteristically  as  rather  small,  round, 
transparent  colonies. 

(b)  The  Enumeration  of  Bacteria. — A  very  practical  application  of 
the  plating  method  for  the  isolation  of  bacteria  is  the  enumeration  of 
bacteria  in  water,  milk  and  other  similar  substances.  The  principle 
involved  depends  upon  the  development  of  colonies  of  bacteria  from 
single  cells.  If  a  definite  volume  of  water,  1  c.c.  for  example,  is  dis- 
tributed in  melted  agar,  thoroughly  mixed  in  the  tube  by  rotation 
between  the  hands,  and  poured  carefully  into  a  sterile  Petri  dish,  the 
number  of  colonies  which  develop  within  a  definite  period  of  incuba- 
tion may  be  regarded  as  a  measure  of  the  number  of  living  bacterial 


216      MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

cells  in  a  cubic  centimeter  of  water.  Experience  has  shown  that  the 
accuracy  of  the  method  is  influenced  somewhat  by  the  number  of 
organisms  in  the  sample.  A  large  number  of  bacteria,  by  mutual 
antagonism,  will  fail  to  develop  into  a  proportionate  number  of  colonies. 
The  most  accurate  results  are  obtained  when  the  bacterial  content  of 
the  sample  as  plated  lies  between  fifty  and  two  hundred  individual 
organisms.  If  more  than  two  hundred  bacteria  are  probably  present, 
a  dilution  of  the  sample  with  sterile  water  is  made  before  plating,  to 
reduce  this  source  of  error.  It  is  convenient  in  making  dilutions  to 
use  a  multiple  of  ten,  because  the  subsequent  calculation  is  much 
simplified.  A  dilution  of  1  to  10  is  made  by  adding  1  c.c.  of  the  sample 
to  9  c.c.  of  sterile  water,  shaking  thoroughly  and  plating  1  c.c.  If 
the  technic  is  all  right,  each  colony  on  the  plate  represents  one-tenth 
the  number  of  living  bacteria  in  the  original  sample.  The  total  number 
of  colonies  multiplied  by  ten  gives  the  theoretical  bacterial  count  of 
the  sample.  A  dilution  1  to  100  is  made  by  adding  1  c.c.  of  the  sample 
to  99  c.c.  of  sterile  water.  The  plating  method  is  inexact,  partly  because 
an  unknown  proportion  of  organisms  in  the  original  sample  will  fail 
to  develop  for  various  reasons  in  the  cultural  medium;  furthermore, 
certain  types  of  organisms,  as  streptococci,  may  remain  adherent 
in  chains  of  greater  or  lesser  length  and  develop  as  a  single  colony. 
Anaerobic  bacteria  do  not  develop  under  aerobic  conditions. 

A  template  of  paper  or  glass  ruled  in  square  centimeters  is  used 
to  facilitate  the  enumeration  of  colonies;  for  densely  colonized  plates, 
each  centimeter  square  of  the  template  is  subdivided  into  smaller 
units,  usually  one-ninth  of  a  square  centimeter.  The  Petri  dish  con- 
taining colonies  is  placed  upon  the  template  in  such  a  manner  that 
the  colonies  appear  superimposed  upon  the  rulings.  It  is  a  simple 
matter,  with  the  lines  as  a  guide,  to  count  either  the  entire  number  of 
colonies  in  the  Petri  dish,  or  a  few  representative  areas,  which  can  be 
multiplied  by  a  factor.  (The  average  Petri  dish  contains  about  63 
square  centimeters.) 

Example. — A  sample  of  milk  diluted  1  to  100  shows  a  large  number 
of  colonies  after  forty-eight  hours'  incubation.  The  total  count  of 
nine  squares  (each  a  square  centimeter)  is  180  colonies,  an  average 
of  twenty  colonies  per  square  centimeter.  The  colonies  upon  the 
entire  plate  (63  square  centimeters)  is  63  x  20,  or  1260.  The  number 
of  living  bacteria  in  1  c.c.  of  the  sample  of  milk  would  be  1260  x  100 
or  126,000,  because  the  number  of  colonies  upon  the  plate  is  T^TT  the 
entire  number  in  1  cm. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      217 

The  value  of  the  method  as  a  convenient  means  of  comparison  of  the 
bacterial  content  of  various  samples  of  milk,  water,  sewage,  and  the 
like  depends  largely  upon  the  supposition  that  the  same  types  of  bac- 
teria present  in  different  samples  will  grow  quantitatively  under  like 
conditions.  The  comparison  of  bacterial  counts  is  therefore  a  com- 
parison of  a  section  of  the  total  bacterial  flora,  not  an  absolute  measure 
of  the  number  of  living  organisms.  The  method  of  counting  bacterial 
colonies  has  been  highly  developed  for  the  regulation  of  water  and  milk 
supplies  of  cities.  (See  section  Water  and  Milk.) 

(c)  Growth  of  Bacteria  in  Gelatin. — Gelatin  is  added  to  cultural 
media  both  to  confer  upon  the  media  the  property  of  solidifying,  and 
to  enrich  the  content  in  nitrogenous  substances. 

Pure  gelatin  does  not  contain  tyrosine  and  it  is  relatively  rich  in 
diamino  acids;  according  to  Hausmann,1  nearly  36  per  cent,  of  the 
nitrogen  in  gelatin  is  diamino  nitrogen — about  63  per  cent,  in  the  form 
of  mono-amino  acids.  Chemically,  gelatin  media  are  convenient  for 
the  demonstration  of  soluble,  proteolytic  enzymes.2  In  the  absence  of 
utilizable  carbohydrate,  several  types  of  bacteria  "liquefy"  gelatin, 
that  is,  through  the  activity  of  their  proteolytic  enzymes  the  gelatin 
molecule  is  split  by  hydrolytic  cleavage  to  molecules  so  simple  in 
their  state  of  aggregation  that  they  can  no  longer  produce  a  "gel." 
The  presence  of  utilizable  carbohydrate  prevents  the  liquefaction  of 
gelatin  by  many  bacteria.3 

Formerly  the  morphology  of  the  liquefied  zone  in  gelatin  stab  cul- 
tures was  regarded  as  distinctive  for  individual  organisms;  thus,  the 
napiform  liquefaction  produced  by  cholera  vibrios  was  supposed  to 
be  sufficiently  constant  to  possess  diagnostic  value.  It  is  now  generally 
conceded  that  this  morphological  characteristic  is  of  comparatively 
little  importance  for  the  identification  of  the  organism.  On  the  other 
hand,  the  liquefaction  of  gelatin  and  of  coagulated  blood  serum  and 
casein  as  well  is  important  from  a  chemical  viewpoint,  because  it 
indicates  the  activity  of  a  soluble  proteolytic  enzyme.4 

II.  Growth  of  Bacteria  in  Fluid  Media. — (a)  Plain  Broth. — Plain 
broth,  prepared  from  meat  infusion  and  peptone  in  the  usual  manner, 

1  Zeit.  f.  physiol.  Chem.,  1899,  xxvii,  95. 

2  Kendall,  Boston  Med.  and  Surg.  Jour.,  1913,  clxviii,  825. 

3  Kendall  and  Walker,  Jour.  Inf.  Dis.,  1915,  xvii,  442. 

.  4  The  enzyme  may  be  obtained  sterile  and  in  an  active  state  in  the  filtrates  of  liquefied 
gelatin,  blood  serum,  casein,  and  from  plain  broth  cultures  as  well,  if  the  bacteria  are 
removed  by  filtration  through  unglazed  porcelain:  Auerbach,  Arch.  f.  Hyg.,  1897, 
xxxi,  311;  Berghaus,  ibid.,  1906,  Ixiv,  1;  Kendall  and  Walker,  Jour.  Inf.  Dis.,  1915, 
xvii,  442. 


218     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

and  freed  from  sugar  with  Bacillus  coli,1  contains,  on  the  average, 
about  300  milligrams  of  nitrogen  per  100  c.c.  A  small  but  variable 
amount  of  the  nitrogen  exists  as  free  ammonia.2  Less  than  10  per 
cent,  of  the  total  nitrogen,  as  a  rule,  exists  as  aminonitrogen  (deter- 
mined by  the  method  of  Van^Slyke)  .3 

The  visible  changes  in  the  appearance  of  broth  cultures  incidental 
to  the  development  of  bacteria  are  not  of  great  importance;  they 
consist  essentially  of  turbidity,  sediment,  and  occasionally  a  ring 
or  pellicle.  The  development  of  a  pellicle  is  of  importance  in  the  pro- 
duction of  toxin  by  the  diphtheria  bacillus,  however,  because  it  indi- 
cates the  maximum  oxygenation  of  the  bacteria.  The  character  of 
the  turbidity  and  sediment — the  viscidity  and  color — may  afford 
some  information  of  the  character  of  the  organism.  Products  of 
importance  are  frequently  detected  by  chemical  or  physiological 
examination  in  plain  broth  cultures  of  bacteria.  Thus,  in  the  absence 
of  utilizable  carbohydrate,  diphtheria  and  tetanus  bacilli  produce 
their  very  potent  toxins;4  proteolytic  bacteria  elaborate  soluble 
enzymes;5  Bacillus  coli,  Bacillus  proteus  and  other  bacteria  form  indol 
and  phenolic  bodies  from  tryptophan  and  tyrosine  respectively;  the 
cholera  vibrios  form  nitroso  indol,6  and  in  sugar-free  broth  containing 
freshly  drawn,  sterile,  defibrinated  blood,  various  bacteria  produce 
hemolysis. 

The  rate  of  decomposition  of  the  protein  constituents  of  the  broth 
may  be  measured  by  the  Folin  microscopic  method  for  ammonia;  the 
increase  in  ammonia  indicates  the  extent  of  deaminization.7  The 
rate  of  hydrolysis  of  protein  is  conveniently  estimated  with  the  Van 
Slyke8  amino-acid  apparatus,  after  removal  of  ammonia  from  the 
medium.9  A  combination  of  these  methods  affords  an  approximate 
analysis  of  plain  broth  media  before  and  after  bacterial  growth.  Un- 
doubtedly the  application  of  the  Emil  Fischer  esterification  method 
of  aminonitrogen  determination  will  throw  much  light  upon  the  utili- 
zation of  various  amino  acids  by  specific  bacteria  during  their  growth 
in  artificial  media.  Amino  acids  containing  aromatic  nuclei — as  tryp- 

1  Theobald  Smith,  Cent.  f.  Bakt.,  1897,  xxii,  45. 

2  Determined  by  the  Folin  Method,  Jour.  Biol.  Chem.,  1912,  xi,  523. 

3  Jour.  Biol.  Chem.,  1912,  xii,  275;  1913,  xvi,  121. 

4  Theobald  Smith,  Tr.  Assn.  Am.  Phys.,  1896;  Jour.  Exp.  Med.,  1899,  iv,  373. 

5  Kendall  and  Walker,  Loc.  cit. 

c  Kendall,  Jour.  Med.  Res.,  1911,  xxv,  117. 

7  Kendall  and  Farmer,  Jour.  Biol.  Chem.,  1912,  xii,  13,  215,  219,  465;  xiii,  63;  Kendall, 
Day  and  Walker,  Jour.  Am.  Chem.  Soc.,  1913,  xxxv,  1201;  1914,  xxxvi,  1937. 

8  Van  Slyke,  Loc.  cit. 

9  The  rate  of  hydrolysis  may  also  be  estimated  by  Sorenson's  formol  titration  method, 
but  this  is  less  accurate  for  small  amounts  than  Van  Slyke's  method. 


METHODS  FOR   THE  MICROSCOPIC  STUDY  OF  BACTERIA      219 

tophan,  tyrosine,  histidin — give  colored  compounds  with  various 
reagents  because  they  contain  the  chromophoric  group,  C  =  C.  The 
formation  of  indol  from  tryptophan  (see  page  74  for  chemistry)  has 
long  been  used  as  a  diagnostic  test  for  Bacillus  coli  and  other  bacteria. 
The  test  depends  upon  the  removal  of  alanin  from  the  tryptophan 
molecule  by  the  activity  of  the  organism,  and  the  addition  of  an  auxo- 
chromic  group,  NO2  in  the  beta  position  of  the  pyrrol  ring,  previously 
occupied  by  alanin.  In  an  acid  medium  the  compound,  betanitroso- 
indol,  is  brownish  red. 

Procedure,  Indol  Test. — To  a  three-day  plain  broth  culture  of  Bacil- 
lus coli  (or  other  organism)  add  1  c.c.  of  concentrated  hydrochloric 
acid.1  Mix  thoroughly  and  overlay  the  acid  broth  with  1  to  2  c.c.  of  a 
0.1  per  cent,  solution  of  sodium  nitrite.2  At  the  junction  of  the  two 
solutions  a  brownish-red  ring  of  nitroso  indol  develops. 

(b)  Carbohydrate  Broths. — The  addition  of  sugars,  as  dextrose,  lac- 
tose, saccharose,  or  of  alcohols,  as  glycerol,  to  plain  broth  media, 
greatly  enriches  the  medium  in  non-nitrogenous  substances  which 
may  be  readily  utilizable  sources  of  energy  for  bacteria.  It  is  hardly 
necessary  to  emphasize  the  importance  of  purity  in  all  sugars  and 
other  carbohydrates  intended  for  bacterial  purposes,  nor  the  fallacy  of 
attempting  to  determine  the  action  of  bacteria  upon  specific  carbohy- 
drates in  media  not  freed  from  muscle  sugar  (dextrose).  The  use  of 
serum  as  a  basis  for  fermentation  media  frequently  introduces  a  source 
of  error,  because  blood  serum  normally  contains  about  0.08  per  cent, 
of  dextrose,  an  amount  quite  sufficient  to  give  rise  to  considerable 
amounts  of  acid.3 

The  observations  made  in  carbohydrate  media  are  usually  restricted  to : 

(a)  Change  in  reaction. 

(b)  Gas  formation,  and  in  fermentation  tubes,  to  growth  in  the 
closed  arm  as  well. 

1  Any  strong  mineral  acid  will  answer  the  purpose. 

2  Best  accomplished  by  running  the  nitrite  solution  carefully  down  the  side  of  the 
tube  held  in  a  slanting  position;  a  stratification  of  the  two  liquids  should  be  obtained. 

3  The  significance  of  fermentation  media  for  the  classification  and  identification  of 
bacteria  depends  upon  their  content  both  of  protein  and  carbohydrate.    Bacteiia  derive 
their  energy  requirements  from  carbohydrate,  if  it  is  utilizable,  but  of  course  they  must 
obtain   their  "Bausteine"    from   the   nitrogenous   constituents.      If   the   carbohydrate 
cannot  be  utilized,  both  structural  and  energy  requirements  are  derived  from  the  protein 
constituents.     Bacteria  vary  greatly  in  their  ability  to  ferment  carbohydrates;  some 
types,  as  Bacillus  alcaligenes,  do  not  appear  to  ferment  even  dextrose.     Bacillus  lactis 
aerogenes,  on  the  contrary,  can  ferment  hexoses,  bioses,  and  even  starches.     The  fer- 
mentability  of  a  carbohydrate  depends  apparently  upon  its  stereo-isomeric  configuration, 
and  relatively  slight  differences  in   the   configuration   of  similar   carbohydrates   may 
determine  their  value  for  specific  organisms  as  sources  of  energy.    This  point  is  discussed 
somewhat  later  in  this  section. 


220     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

Bacteria  which  can  utilize  carbohydrates  for  their  energy  require- 
ments usually  produce  acid;  many  types  produce  gas  as  well.  The 
acid,  which  is  commonly  lactic,  together  with  small  amounts  of  acetic 
and  other  fatty  acids  may  be  estimated  by  titration  with  standard 
alkali.  A  more  accurate  estimation  is  based  upon  the  determination 
of  the  hydrogen  ion  concentration.1  The  gases  formed  are  usually 
carbon  dioxide  and  hydrogen.  An  approximate  ratio  of  the  proportion 
H/CO2  is  conveniently  made  in  the  Smith  Fermentation  Tube,2  in  the 
following  manner: 

The  level  of  the  gas  in  the  closed  arm  is  marked  with  a  wax  pencil. 
The  bulb  of  the  fermentation  tube  is  then  completely  filled  with 
a  1  per  cent,  solution  of  sodium  solution,  and  the  gas  brought 
into  contact  with  the  alkaline  solution  by  inverting  the  tube  several 
times.  The  gas  is  then  entirely  run  back  into  the  closed  arm,  and  the 
volume  again  measured.  The  volume  is  diminished  proportionately 
to  the  absorption  of  CO2  by  the  caustic  alkali. 

Smith3  has  determined  the  "gas  ratio"  for  the  principal  aerogenic 
bacteria  as  follows : 

Organism.  Dextrose.  Lactose.  Saccharose 

H         C02  H          CO2  H         C02 

B.  coli 63  37  63  37  63  37 

Hog  cholera 66  34 

B.  lactis  aerogenes  ....  65  35  62  38  80  20 

Friediander  bacillus      ...  67  33  86  14  67  33 

B.  edematis  maligni      ...  67  33  ?  . .  ? 

B.  proteus 72  28  67  33 

B.  cloacse  70  30  37  63  58  42 


Bacteria  -which  ferment  sugars  grow  in  the  closed  arm  of  the  fer- 
mentation tube;  those  organisms,  with  very  few  exceptions,  which 
cannot  utilize  the  carbohydrate  of  a  fermentation  medium  fail  to 
grow  in  the  closed  arm  where  free  oxygen  is  not  available;  growth 
appears  only  in  the  open  arm. 

Occasionally  a  very  slight  change  in  the  stereo-isomeric  formula  of 
a  carbohydrate,  or  a  very  small  change  in  its  terminal  groups  will 
determine  its  fermentability  by  various  organisms.  Thus  dextrose, 
mannose,  and  their  respective  alcohols,  sorbite  and  mannite,  according 
to  Emil  Fischer,4  have  the  following  stereo-isomeric  formulae: 

1  Clark,  Jour.  Inf.  Dis.,  1915,  xvii,  109. 

2  Theobald  Smith,  The  Fermentation  Tube,  Wilder  Quarter  Century  Book,   1895, 
187  et  seq. 

3  LOG.  cit. 

4  Untersuchungen  iiber  Kohlenhydrate  und  Fermente,  1884-1908,  Berlin,  1902. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      221 

H  H 

I  I 

H— C=O                           H— C=O                          H— C— OH  H— C— OH 

I                                           I                                          I  I 

H— C— OH                     HO— C— H                          H— C— OH  HO— C— H 

I                                           I                                           I  I 

HO— C— H                        HO— C— H                       HO— C— H  HO— C— H 

till 

H— C— OH                        H— C— OH                       H— C— OH  H— C— OH 

I                                           I                                           I  I 

H— C— OH                        H— C— OH                       H— C— OH  H— C— OH 

I                                           I                                           I  I 

H— C— H                          H— C— H                          H— C— H  H— C— H 

I                                            I                                            I  I 

o                             o                             o  o 

H                                        H                                        H  H 

D.  Glucose.                     D.  Mannose.                     D.  Sorbite.  D.  Mannite 


The  fermentation  of  these  hexoses  and  their  respective  alcohols 
by  certain  bacteria  is  shown  in  the  accompanying  table: 

Organism.  D.  Dextrose  D.  Mannose.  D.  Sorbite.  D.  Mannite 

B.  dysenterise  Shiga  +  + 

B.  dysenteiise  Flexner  .      +  +  + 

B.  Morgan  No.  1   .      .  .      +  + 

B.  paratyphosus  Beta  +  +  +  + 

An  explanation  for  the  phenomenon  set  forth  in  the  table  does  not 
readily  suggest  itself.  Somewhat  similar  selective  action  upon  specific 
amino  acids  by  other  bacteria  is  known,  qualitatively  at  least.  Thus, 
members  of  the  Hemorrhagic  Septicemia  Group,  particularly  those 
derived  from  animal  sources,  produce  indol  in  plain  broth  media. 
Typhoid  bacilli,  diphtheria  bacilli  and  many  other  pathogenic  organ- 
isms usually  fail  to  produce  indol  in  ordinary  media  under  similar  con- 
ditions. It  is  possible  that  this  noteworthy  action  of  members  of  the 
Hemorrhagic  Septicemia  Group  upon  tryptophan  may  be  related  to 
the  fact  that  this  amino  acid  is  an  important  constituent  of  the  hemo- 
globin, the  coloring  matter  of  the  blood;  the  Hemorrhagic  Septicemia 
Bacteria  are  particularly  likely  to  grow  in  the  blood  stream  of  infected 
animals. 

Fermentation  reactions  of  bacteria  in  varied  carbohydrate  media 
are  of  importance  in  their  cultural  identification.  The  table  on  page 
316  illustrates  the  separation  of  members  of  the  Intestinal  Group  of 
Bacteria  by  their  fermentation  reaction  in  various  carbohydrates. 

Milk. — Milk  is  an  important  natural  medium  for  bacterial  growth. 
It  contains  protein,  carbohydrate  and  fat,  together  with  inorganic 
salts.  A  variety  of  reactions  and  changes  in  milk  are  produced  by 
bacterial  development. 


222     MICROSCOPIC  AND  CULTURAL  STUDY  OF  BACTERIA 

(a)  Change  in  Reaction. — Milk  contains,  in  addition  to  protein,  two 
carbohydrates,  which  play  a  prominent  part  in  determining  the  reac- 
tion of  the  medium.  The  principal  carbohydrate  is  lactose,  but  fresh 
milk  contains  in  addition,  a  small  amount — about  0.08  per  cent. — of  a 
sugar  reacting  like  dextrose.1  Changes  in  the  reaction  of  milk  caused 
by  bacterial  activity,  therefore,  may  be  of  several  types.  An  initial 
acidity  followed  by  an  alkaline  reaction,  as  exhibited  by  the  dysentery 
bacilli  and  other  organisms,  is  probably  due  to  the  initial  fermentation 
of  the  small  amount  of  dextrose,  which  results  in  the  formation  of 
acid — and  then  the  production  of  alkaline  products  from  the  decom- 
position of  protein  when  the  dextrose  is  exhausted.  These  organisms 
do  not  ferment  lactose. 

A  permanent  acid  reaction  is  induced  either  by  bacteria  which  fer- 
ment lactose,  or  less  commonly,  by  the  decomposition  of  fat  with  the 
liberation  of  fatty  acids.  Bacillus  typhosus  and  Bacillus  paratyphosus 
alpha  produce  a  permanently  acid  reaction  in  milk,  but  do  not  ferment 
lactose.  The  exact  chemistry  of  their  activity  in  the  medium  is  not 
known.  An  alkaline  reaction  in  milk  is  usually  an  indication  of  proteo- 
lytic  action  with  the  formation  of  basic  products  of  protein  decom- 
position. 

The  accumulation  of  acid  incidental  to  the  fermentation  of  lactose, 
as,  for  example,  by  B.  coli,  may  be  sufficient  in  amount  to  cause  an 
acid  coagulation  of  the  casein.2  An  acid  coagulation  can  be  distin- 
guished from  an  enzyme  (lab  or  rennin)  coagulation;  the  acid  coagulum 
will  redissolve  in  alkali,  but  an  enzyme  coagulum  fails  to  redissolve 
by  merely  neutralizing  the  reaction. 

Some  types  of  bacteria,  as  Bacillus  aerogenes  capsulatus,  ferment 
lactose  energetically,  liberating  a  large  amount  of  gas,  and  forming 
butyric  acid  as  well.  For  some  unknown  reason,  Bacillus  coli  and 
allied  organisms,  which  ferment  lactose  in  fermentation  tubes  with 
the  liberation  of  considerable  amounts  of  gas,  fail  to  produce  gas  from 
the  lactose  as  it  exists  in  milk.  It  has  been  shown,  however,3  that  the 
colon  bacillus  will  liberate  gas  from  lactose  if  the  milk  is  first  acted  upon 
by  a  strongly  proteolytic  organism,  as  B.  mesentericus. 

Proteolytic  bacteria,  which  are  unable  to  utilize  either  the  small 
amount  of  dextrose,  the  lactose  or  the  fats  of  milk,  usually  produce 

1  Theobald  Smith,  Jour.  Boston  Soc.  Med.  Sci.,  1897,  ii,  236;  Jones,  Jour.  Inf.  Dis., 
1914,  xv,  357. 

2  It  must  be  remembered  that  bacteria  grown  in  litmus  milk  frequently  fail  to  cause 
coagulation  unless  the  medium  is  heated  to  boiling. 

3  Kendall,  Boston  Med.  and  Surg.  Jour.,  1910,  clxiii,  322. 


METHODS  FOR  THE  MICROSCOPIC  STUDY  OF  BACTERIA      223 

an  alkaline  reaction  which  may  be  a  simple  alkalinity  without  obvious 
change  in  the  appearance  of  the  medium  (as,  for  example,  B.  alkali- 
genes)  or  a  deep  peptonization  of  the  casein,  as  illustrated  by  B. 
pyocyaneus.  B.  mesentericus  peptonizes  casein  energetically,  but 
the  reaction  of  the  medium  is  persistently  acid.  The  initial  acidity 
is  probably  due  to  the  formation  of  acid  from  the  dextrose  of  the 
milk;  the  residual  acid  may  be  associated  with  the  activity  of  an 
esterase  which  certain  strains  of  this  organism  appear  to  elaborate. 
Fatty  acids  are  formed  by  hydrolysis  of  the  glycerides  of  the  cream 
by  the  soluble  esterase,  while  the  metabolic  activities  of  the  organism 
appear  to  be  largely  directed  to  the  proteins  of  the  medium.1 

It  is  apparent,  therefore,  that  the  chemical  and  physical  changes 
induced  in  milk  incidental  to  bacterial  development  in  the  medium 
are,  or  may  be,  complex  in  their  origin.  A  knowledge  of  the  proteo- 
lytic  and  fermentative  activities  of  bacteria  in  the  simpler  media, 
however,  will  frequently  furnish  an  explanation  for  the  more  involved 
reactions  in  the  highly  complex  medium,  milk. 

1  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Soc.,  1914,  xxxvi,  1937. 


CHAPTER  X. 


BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL  FROM 
THE  PATIENT  AND  THE  CADAVER. 


MATERIAL  FROM  THE  LIVING  SUBJECT. 
Blood  Culture. 

Technic  of  Blood  Cultures. 
Bacteriological  Examination  of  Cere- 

brospinal  Fluid. 
The  Examination  of  Peritoneal,  Pleu- 

ral  and  Pericardial  Fluids. 
Pus. 

Examination  of  Urine. 
Examination  of  Feces. 
Examination  of  Sputum,  of  Buccal 

and  Pharyngeal  Material. 


Examination  by  Staining. 
Cultural  Methods. 

Bacteriological    Examination    of    the 
Eye. 

Bacteriological    Examination    of    the 

Ear  and  Nose. 

THE  UTILIZATION  OF  ANIMALS  FOR  BAC- 
TERIAL DIAGNOSIS  AND  EXPERI- 
MENTATION. 

The  Inoculation  of  Animals. 


THE  successful  outcome  of  a  bacteriological  examination  of  material 
from  a  patient  or  a  cadaver  depends  to  a  large  degree  upon  the  appli- 
cation of  proper  technic  at  the  time  of  collection.  Naturally  this  is 
varied  according  to  the  nature  of  the  case. 

Postmortem  cultures  are  taken  from  organs  or  tissues  usually 
indicated  by  the  nature  of  the  infection,  and  a  choice  of  media  for  the 
isolation  of  a  specific  bacteria,  or  types  of  bacteria,  is  made  with  this 
information  in  view.  The  value  of  a  postmortem  bacteriological 
examination  is  frequently  measured  by  the  promptness  with  which 
it  is  made  after  death;  postmortem  invasion  of  tissues,  organs,  and 
even  the  heart  and  larger  bloodvessels  by  bacteria  from  the  mouth 
and  gastro-intestinal  tract  takes  place  very  quickly.  Even  if  the 
cadaver  is  placed  at  once  in  a  cold  room,  some  time  must  elapse  before 
the  internal  organs  are  cooled  sufficiently  to  arrest  bacterial  growth. 

The  spleen,  liver,  kidneys,  and  bloodvessels  are  more  commonly 
examined  for  evidence  of  pathogenic  microorganisms.  The  surface 
of  the  undisturbed  organ  is  first  seared  with  a  hot  iron,  then  incised 
through  the  sterile  area,  and  some  of  the  contents  withdrawn  in  a 
platinum  loop  or  with  a  sterile  capillary  pipette  and  introduced  at  once 
into  suitable  media.  (The  kind  of  media  to  be  used  is  clearly  set  forth 
for  each  organism,  in  succeeding  chapters.)  Blood  may  be  obtained 
from  the  heart,  after  searing  the  surface  of  the  organ,  or  from  the 
larger  veins  of  the  extremities.  Exudates  from  the  pleural,  peritoneal 


MATERIAL  FROM  THE  LIVING  SUBJECT  225 

or  pericardial  cavities  may  be  removed  with  sterile  pipettes  and  trans- 
ferred temporarily  to  sterile  test-tubes  or  flasks.  Purulent  discharges 
are,  if  small  in  amount  aspirated  directly  into  sterile  capillary  pipettes ; 
if  in  considerable  quantity,  removed  to  test  tubes  or  flasks,  and  inocu- 
lated as  soon  as  practicable  into  suitable  media. 

MATERIAL  FROM   THE  LIVING   SUBJECT. 

Blood  Cultures. — The  organisms  of  septicemia  may  be  numerous 
or  few  in  number  in  the  blood  stream — furthermore,  they  may  be 
associated  with  specific  lysins  and  agglutinins,  as  occasionally  hap- 
pens in  typhoid  fever.  For  these  various  reasons,  experience  has  shown 
that  from  5  to  15  c.c.  of  blood,  drawn  aseptically,  should  be  discharged 
at  once  with  aseptic  precautions,  into  at  least  100  c.c.  of  0.1  per  cent, 
meat  infusion  dextrose  broth,1  and  evenly  distributed  by  careful  agita- 
tion. The  degree  of  dilution  attained  practically  renders  lytic  action 
and  agglutination  ineffective;  the  enrichment  of  the  medium  by  the 
relatively  large  proportion  of  blood  creates  a  very  favorable  medium 
for  the  development  of  the  organisms. 

Technic  of  Blood  Cultures. — 1.  Apparatus. — An  all-glass  syringe  with 
a  platinum-iridium  needle  of  moderately  large  bore  is  sterilized  in  the 
autoclave,  preferably  enclosed  in  a  large  test  tube.  A  syringe  cannot 
be  sterilized  for  bacterial  purposes  by  boiling  in  water. 

As  an  alternate  apparatus,  a  250  c.c.  Ehrlenmeyer  flask  fitted  with 
a  rubber  stopper  containing  two  glass  tubes  bent  at  right  angles  may 
be  used.  The  flask  contains  100  c.c.  of  0.1  dextrose  meat  infusion 
broth.  One  tube  is  connected  with  a  platinum-iridium  needle  by  a 
short  length  of  rubber  tubing,  and  the  needle  is  protected  during  steril- 
ization by  a  small  test  tube  slipped  over  it  and  extending  its  full 
length.  The  test  tube  is  removed  when  the  blood  is  to  be  taken.  The 
other  tube  is  protected  by  a  short  length  of  rubber  tubing  containing 
a  small  filter  of  absorbent  cotton.  Suction  is  applied  through  the 
latter  tube.  It  will  be  seen  that  blood  may  be  drawn  directly  into 
the  broth  in  this  apparatus,  and  in  practice  it  has  been  found  con- 
venient to  replace  the  rubber  stopper  with  a  sterile  cotton  plug  after 
the  blood  is  mixed  with  the  media. 

2.  Collection  of  Blood. — The  skin  over  the  median  basilic  vein  is 
cleansed  with  green  soap  and  alcohol,  dried,  and  sterilized  by  the 
application  of  tincture  of  iodine,  which  is  allowed  to  act  for  two  to 

i  See  Media. 
15 


22(5         BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL 

three  minutes.  Then  the  point  of  the  needle  is  gently  inserted  into 
the  vein  (which  may  be  made  prominent  by  gentle  pressure  with  a 
tourniquet  applied  to  the  arm  above  the  elbow),  and  from  5  to  20  c.c. 
of  blood  withdrawn.  This  is  introduced  at  once  into  broth,  as  outlined 
above.1 

It  may  be  desirable  to  estimate  the  number  of  bacteria  in  the  blood : 
1  c.c.  of  blood  is  mixed  at  once  with  10  c.c.  of  agar  previously  melted 
and  cooled  to  42°  C.,  and  plated  in  a  Petri  dish.  If  desired,  dilution 
may  be  made  1  to  10,  1  to  100  in  succeeding  tubes  of  agar. 

Typhoid  and  paratyphoid  bacilli  grow  readily  in  the  broth  cultures. 
They  may  be  identified  by  their  cultural  and  agglutination  reactions 
with  highly  potent  specific  sera.  Streptococcus  and  pneumococcus 
cultures  are  obtained  in  a  similar  manner  from  the  blood  stream  in 
blood  bouillon.  The  organisms  are  isolated  in  pure  culture  by  smear- 
ing the  broth,  after  incubation,  upon  the  surface  of  freshly  prepared 
blood  agar  plates.  The  Streptococcus  colonies  usually  exhibit  a  wide 
clear  zone  of  hemolysis.  Pneumococcus  colonies  are  characterized  by 
a  narrower  green  zone  of  altered  blood  pigment  around  them.  Plague 
bacilli  and  Micrococcus  melitensis  are  frequently  detected  in  the 
blood  stream;  occasionally  the  organisms  are  present  in  sufficient 
numbers  to  develop  in  blood  agar  plates  inoculated  with  1  to  2  c.c. 
of  blood.  The  former  produces  characteristic  lesions  in  guinea-pigs; 
the  latter  develops  very  slowly,  frequently  becoming  visible  only  after 
five  to  seven  days'  incubation. 

Bacteriological  Examination  of  Cerebrospinal  Fluid. — Spinal  fluid 
for  bacteriological  examination  is  obtained  by  lumbar  puncture  with 
a  sterile  hypodermic  needle,  or  fine  trochar  about  8  cm.  long  and  1 
mm.  in  bore.  The  needle  is  introduced  preferably  in  the  fourth  intra- 
vertebral  space;  the  fasciculi  of  the  cauda  equinum  are  not  tense  at 
this  level  and  are  readily  pushed  aside  by  the  needle  without  injury. 
An  imaginary  line  touching  the  crests  of  the  ilia  intersects  the  spinous 
process  of  the  fourth  lumbar  vertebra;  the  sterile  needle  is  inserted 
through  the  previously  sterilized  skin  at  a  point  1  cm.  to  the  right 
(or  left)  of  the  lower  rim  of  the  spinous  process,  and  directed  obliquely 
upward  and  inward  to  such  a  degree  that  the  point  of  the  needle  will 
reach  the  median  line  at  a  depth  of  5  to  6  cm.  The  subarachnoid  space 
is  reached  at  this  level  and  resistance  to  the  passage  of  the  needle 

1  Occasionally  circumstances  arise  which  make  it  necessary  to  send  the  blood  to  a 
distance  for  examination;  mixing  the  blood  with  an  equal  volume  of  glycerine  bile  (one 
part  glycerin,  ten  parts  ox  bile;  sterilize  in  autoclave)  is  said  to  be  an  efficient  method 
for  preserving  the  bacterial  content  of  blood  practically  unchanged  for  several  hours. 


MATERIAL  FROM   THE  LIVING  SUBJECT  227 

ceases,  and  spinal  fluid  should  flow  at  once.  The  fluid  should  be  col- 
lected in  a  sterile  test  tube.  Usually  from  20  to  30  c.c.  of  fluid  flow 
spontaneously;  the  flow  may  be  much  greater,  75  c.c.  or  even  more. 
Rarely  but  a  few  drops,  or  even  none  at  all  may  be  obtained.  Normal 
spinal  .fluid  is  clear  and  practically  colorless.  Only  a  few  cells,  chiefly 
lymphocytes,  may  be  found  in  the  sediment  obtained  by  centrifugaliza- 
tion.  Pathologically  the  fluid  may  contain  numerous  cellular  elements. 
A  blood-stained  spinal  fluid  may  be  due  to  injury  to  bloodvessels  dur- 
ing the  passage  of  the  needle,  or  to  blood  from  hemorrhage  in  the  brain 
or  upper  levels  of  the  cord.  In  the  former  case  the  blood  will  clot  if 
the  spinal  fluid  is  allowed  to  stand;  in  the  latter  case  the  blood  settles 
to  the  bottom,  but  fails  to  clot.  A  turbid  spinal  fluid  is  indicative  of 
an  inflammatory  process  in  the  cerebrospinal  axis.  If  the  turbidity 
is  uniform,  pus  cells  are  almost  invariably  present.  Occasionally  the 
fluid  appears  clear,  but  upon  standing,  solitary,  cobweb-like  coagula 
appear,  which  enmesh  cellular  elements  and  bacteria  that  may  be 
present.  Sometimes  an  artificial  stimulus  to  coagulation  is  produced 
by  adding  a  fibre  or  two  of  sterile  cotton. 

The  spinal  fluid  should  be  centrifugalized  and  some  of  the  sediment 
stained  with  Wright's  stain  to  determine  the  types  of  leukocytes  and 
organisms  present.  Polymorphonuclear  leukocytes  indicate  an  infec- 
tion with  meningococcus,  parameningococcus,  streptococcus,  staphy- 
lococcus,  typhoid,  colon,  influenza  or  plague  bacilli.  The  fluid  is 
usually  more  or  less  turbid.  Tubercular  infection,  which,  next  to 
meningococcus  infection,  is  the  most  common,  is  usually  accompanied 
by  a  clear  spinal  fluid  from  which  the  cobweb  coagula  mentioned  above 
may  be  obtained  upon  standing.  About  75  per  cent,  of  cases  of  tuber- 
cular meningitis  may  be  diagnosed  through  the  recognition  of  acid- 
fast  bacilli  in  the  stained  smears  from  these  coagula.  It  is  essential, 
in  doubtful  cases,  to  inject  1  to  2  c.c.  of  spinal  fluid  subcutaneously 
into  guinea-pigs.  If  the  inguinal  glands  are  injured  mechanically  by 
squeezing  them  between  thumb  and  index  finger  before  the  injection 
is  made,  and  the  material  is  introduced  as  near  the  glands  as  possible, 
a  definite  diagnosis  of  tuberculosis  may  frequently  be  made  within 
two  weeks;  ordinarily  four  to  six  weeks  are  required  for  the  develop- 
ment of  tuberculosis  in  the  guinea-pig. 

For  the  diagnosis  of  acute  infections  of  the  cerebrospinal  axis,  about 
10  c.c.  of  spinal  fluid  should  be  withdrawn  with  aseptic  precautions 
into  a  sterile  test  tube.  If  this  fluid  is  visibly  turbid,  direct  smears 
stained  by  Gram's  stain  and  with  Wright's  method  will  furnish  valuable 


228         BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL 

evidence  of  the  etiological  organism,  and  will  indicate  the  medium  to 
use  for  its  isolation  and  identification.  Blood  agar  is  best  suited  for 
the  meningococcus,  parameningococcus,  streptococcus  and  influenza 
bacillus.  The  staphylococcus,  typhoid,  colon  and  plague  bacilli  are 
less  fastidious  in  their  requirements.  Less  commonly,  bacteria  other 
than  those  described  above  are  found  in  the  cerebrospinal  fluid  follow- 
ing infection  of  the  sinuses,  otitis  media,  mastoid  infection  or  septi- 
cemia.  The  virus  of  anterior  poliomyelitis  is  also  found  in  the  spinal 
fluid.  The  most  practical  method  of  diagnosis  for  the  latter  is  to  filter 
the  clear  spinal  fluid  through  a  Berkefeld  filter  to  eliminate  all  bac- 
teria, and  to  inject  5  to  10  c.c.  of  the  filtrate  intraspinously  into 
monkeys.  The  animal  usually  will  exhibit  symptoms  within  two  weeks 
if  the  virus  is  present. 

The  Examination  of  Peritoneal,  Pleural  and  Pericardial  Fluids.— 
Fluids  or  exudations  from  the  peritoneum,  pericardium  or  pleurae 
should  be  stained  by  Gram's  method  to  determine  the  type  of  organism, 
and  by  Wright's  method  to  distinguish  the  types  of  cellular  elements 
and  their  relation  to  the  microorganisms.  If  the  fluid  is  clear,  or  if 
lymphoid  cells  predominate,  an  infection  with  the  tubercle  bacillus  is 
immediately  suggested.  Sediment  from  such  a  fluid  should  be  injected 
into  a  guinea-pig,  using  the  method  outlined  for  suspected  spinal  fluid. 
A  turbid  fluid  usually  indicates  an  infection  with  the  streptococcus, 
pneumococcus,  staphylococcus  or  pneumobacillus,  if  the  material  is 
from  the  pleura?  or  pericardium;  an  infection  with  the  streptococcus 
or  members  of  the  intestinal  group  if  the  source  is  the  peritoneal  cavity. 
Rarely  the  gonococcus  has  been  found.  An  examination  of  the  Gram- 
stained  smear  will  indicate  the  proper  medium  to  use  for  the  isolation 
of  the  organisms  in  pure  culture. 

Pus. — A  Gram  stain  of  pus  will  indicate,  as  a  rule,  the  proper  medium 
to  use  for  the  isolation  and  identification  of  the  organisms.  Pus  from 
'*  cold"  abscesses  frequently  contains  no  organisms  recognizable  either 
by  Gram  or  acid-fast  stains;  experience  has  clearly  demonstrated, 
however,  that  a  small  amount  of  the  material  injected  subcutaneously 
into  guinea-pigs  will  cause  their  death,  frequently  within  three  weeks. 
At  autopsy,  tubercles  and  tubercle  bacilli  are  found  in  abundance. 
Much  and  others  believe  that  tubercle  bacilli  found  in  the  pus  from 
cold  abscesses  do  not  exist  in  their  normal  form,  but  appear  as  gran- 
ules— the  so-called  Much  granules — which  are,  however,  viable  and 
virulent  for  guinea-pigs.  In  this  animal  the  organisms  regain  their 
normal  morphology  and  staining  reactions.  The  possibility  of  Hypho- 


MATERIAL  FROM  THE  LIVING  SUBJECT  229 

mycetes  in  the  pus  from  old  cavities  in  the  lungs  should  be  borne  in 
mind.  Aetinomyces  are  usually  visible  to  the  naked  eye  as  minute,  yel- 
lowish granules  which  exhibit  the  characteristic  club  when  viewed  under 
the  microscope  in  properly  stained  specimens.  Pus  from  abscesses 
in  the  cervical  region  may  contain  spiral  organisms.  The  occurrence 
of  these  organisms  should  suggest  the  possibility  of  a  sinus  connecting 
the  abscess  with  the  mouth.  Frequently  such  a  sinus  originates  at 
the  base  of  a  carious  tooth. 

Examination  of  Urine. — A  bacteriological  examination  of  the 
urine  is  of  value  not  only  in  the  diagnosis  of  infection  of  the  genito- 
urinary system;  it  may  afford  information  of  the  causative  organisms 
in  septicemia,  and  occasionally  those  concerned  in  the  more  chronic 
heart  or  joint  lesions  as  well. 

The  external  genitalia  are  usually  contaminated  with  B.  smegmatis, 
which  resembles  the  tubercle  bacillus,  and  with  various  adventitious 
organisms  as  well.  Prominent  among  the  latter  is  Bacillus  coli.  A 
satisfactory  sample  of  urine  for  bacteriological  examination  may  be 
obtained  from  males  if  the  glans  and  meatus  are  thoroughly  cleansed 
with  soap  and  water.  The  greater  amount  of  urine  passed  should  be 
rejected,  and  the  last  portion  should  be  collected  in  a  sterile,  wide- 
mouthed  bottle.  It  is  necessary  to  catheterize  females  after  a  pre- 
liminary cleansing  with  soap  and  water,  to  obtain  a  satisfactory 
specimen  for  bacteriological  examination.  A  sterile  catheter  must  be 
used,  and  the  first  portion  of  the  urine  should  be  discarded.  Under 
ordinary  conditions,  except  in  tubercle  infections  the  causative 
organisms  will  be  present  in  sufficient  numbers  so  that  a  direct  smear 
of  the  sediment,  stained  by  Gram's  method,  will  furnish  a  valuable 
clue  to  the  method  and  media  to  be  used  for  the  isolation  and  identifi- 
cation of  the  organism. 

Blood  agar  is  a  favorable  medium  for  the  isolation  of  the  streptococ- 
cus, pneumococcus,  gonococcus  and  staphylococcus.  The  gonococcus 
is  usually  recognized  by  a  Gram-stained  smear  without  further  attempt 
at  isolation.  It  is  a  Gram-negative  diplococcus  which,  in  acute  infec- 
tion, usually  appears  both  intra-  and  extracellularly  among  polymor- 
phonuclear  leukocytes.  Micrococcus  catarrhalis,  which  might  easily 
.  be  confused  with  the  gonococcus,  occurs  very  rarely  in  genito-urinary 
infections;  ordinarily  it  may  be  disregarded.  Micrococcus  melitensis 
grows  very  slowly  upon  ordinary  media.  Its  very  small  size  together 
with  the  deliberateness  of  growth  usually  suffice  to  attract  attention 
to  its  presence.  An  agglutination  with  a  specific  serum  completes  the 


230         BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL 

diagnosis.  Streptococci  and  pneumococci  produce  distinctive  changes 
in  the  hemoglobin  of  blood  a'gar  plates.  Their  final  identification 
is  discussed  in  the  section  devoted  to  these  organisms.  Bacillus  coli 
and  Bacillus  proteus  are  common  incitants  of  cystitis;  they  grow  readily 
upon  ordinary  media  and  their  recognition  depends  upon  the  changes 
pure  cultures  induce  in  artificial  media.  (See  table,  page  316.) 

Bacillus  typhosus  and  members  of  the  Paratyphoid  Group  are  occa- 
sionally found  in  the  urine  of  patients  and  convalescents.  The  organ- 
isms are  readily  obtained  in  pure  culture  by  plating  upon  nutrient 
agar,  or,  better,  upon  Endo  medium  (see  page  201).  Their  cultural 
characteristics  and  agglutination  with  specific  sera  establish  their 
identity.  Tubercle  bacilli  may  be  found  in  the  urine;  the  only  satis- 
factory and  trustworthy  diagnosis  is  made  by  injecting  the  sediment 
of  a  twenty-four-hour  sample  of  urine  subcutaneously  into  a  guinea- 
pig.  The  animal  will  succumb  to  infection  if  tubercle  bacilli  are 
present,  but  will  fail  to  react  to  smegma  bacilli,  which  are  acid-fast 
and  resemble  tubercle  bacilli  morphologically. 

Examination  of  Feces. — (See  also  Special  Section,  Bacteriology  of 
the  Feces.) — The  isolation  and  identification  of  pathogenic  microorgan- 
isms from  the  feces  is  frequently  a  difficult  task  because  the  normal 
intestinal  bacteria  preponderate  even  in  severe  infections.  Never- 
theless, the  use  of  special  media  has  greatly  reduced  the  difficulties 
and  a  search  for  specific  microorganisms  is  now  possible  with  a  very 
favorable  outlook  for  success. 

For  convenience,  intestinal  infections  may  be  divided  into  those 
caused  by  cocci,  by  bacilli,  and  spiral  organisms.  Of  the  spherical 
organisms  or  cocci,  the  streptococcus  is  by  far  the  most  common 
pathogenic  organism  encountered  in  intestinal  infections,  although  an 
overgrowth  of  Micrococcus  ovalis  may  be  associated  with  a  distinct 
symptomatology.  The  streptococcus  is  a  common  inhabitant  of  the 
intestinal  tract,  and  for  this  reason  streptococcus  infection  of  the 
alimentary  canal  is  denied  by  many  observers.  The  streptococcus  is 
frequently  an  important  secondary  invader  of  the  intestinal  mucosa 
in  bacillary  dysentery,  and  possibly  in  typhoid  and  paratyphoid  infec- 
tions as  well.  It  is  also  frequently  associated  with  an  overgrowth  of 
the  ''gas  bacillus'5  (Bacillus  aerogenes  capsulatus)  in  intestinal  infec- 
tion with  the  latter  organism.  The  occasional  acute  enteritis  observed 
both  sporadically  and  epidemically  among  young  children  is  also 
incited  by  streptococci.  The  distinction,  if  any  exists,  between  the 
intestinal  streptococcus  and  Streptococcus  pyogenes  is  not  clearly 


MATERIAL  FROM  THE  LIVING  SUBJECT  231 

established.  The  isolation  of  streptococci  from  intestinal  contents 
is  made  either  by  direct  plating  upon  dextrose  agar,  or  by  inoculation 
of  feces  into  dextrose  broth.  The  streptococcus,  as  a  general  rule, 
produces  enough  acid  in  the  medium  after  one  or  two  days'  growth 
at  body  temperature  to  seriously  restrain  the  development  of  the 
intestinal  bacteria.  A  Gram  stain  prepared  from  the  sediment  of  the 
fermentation  tube  will  frequently  reveal  a  nearly  pure  culture  of  the 
organism.  A  direct  smear  from  the  feces,  stained  by  Gram's  method, 
also  will  indicate  the  unusual  preponderance  of  streptococci  in  acute 
streptococcus  enteritis. 

The  members  of  the  alcaligenes,  dysentery,  typhoid,  paratyphoid 
group — comprise  the  more  important  bacilli  ordinarily  sought  for  in 
the  intestinal  contents.  Their  isolation  upon  ordinary  media  is  diffi- 
cult because  Bacillus  coli,  the  most  important  of  the  intestinal  organ- 
isms, greatly  outnumbers  the  more  delicate  pathogenic  bacteria;  its 
colonies  on  ordinary  media  are  not  readily  distinguished  from  typhoid 
colonies.  The  Endo  medium  (see  page  201)  however,  affords  a  ready 
means  of  identification  between  the  pathogenic  bacteria  and  Bacillus 
coli.  The  Endo  medium  is  essentially  lactose  agar  containing  a  small 
amount  of  basic  fuchsin  decolorized  with  sodium  sulphite.  Organic 
acids  including  lactic  acid  restore  the  color  to  fuchsin.  None  of  the 
members  of  the  Alcaligenes-typhoid  Group  ferment  lactose,  therefore 
no  lactic  acid  is  formed  in  and  around  colonies  of  these  bacilli.  Bacillus 
coli,  on  the  other  hand,  ferments  lactose,  and  consequently  the  colonies 
of  this  organism  are  colored  red.  The  lactic  acid  resulting  from  the 
fermentation  of  the  lactose  locally  restores  the  color  to  the  fuchsin. 

Procedure. — A  thin  suspension  in  plain  broth,  prepared  from  a  freshly 
passed  specimen  of  feces,  is  incubated  if  possible,  for  an  hour  at  37° 
C.,  then  rubbed  gently  over  the  surface  of  an  Endo  plate  with  a  sterile 
bent-glass  rod  or  platinum  needle.  At  the  end  of  eighteen  to  twenty- 
four  hours,  small  colorless  transparent  colonies  are  removed  to  0.1 
per  cent,  dextrose  meat  infusion  broth  for  further  development. 
Inasmuch  as  colonies  of  B.  alcaligenes,  dysenterise  (Flexner,  Shiga 
and  other  strains)  typhosus,  paratyphosus  alpha  and  beta,  and  the 
Morgan  bacillus  are  practically  identical  in  appearance,  a  final  iden- 
tification must  depend  upon  their  cultural  characteristics  (see  page 
316  for  table)  and  their  agglutination  with  specific  sera  of  high  potency. 

Members  of  the  Mucosus  Capsulatus  Group  are  occasionally  found 
in  acute  and  subacute  diarrheas.  They  grow  readily  upon  the  surface 
of  Endo  plates  as  very  viscid,  slimy  colonies  which  are  readily  recog- 


2:52         BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL 

nized  by  their  macroscopic  appearance.  Bacillus  pyocyaneus  is  an 
occasional  incitant  of  intestinal  disturbance.  Its  colonies  upon 
ordinary  agar  are  surrounded  by  a  yellowish  or  greenish  halo.  The 
same  general  appearance  characterizes  its  growth  upon  Endo  medium. 
Among  the  anaerobic  bacilli,  the  'gas  bacillus"  (Bacillus  aerogenes 
capsulatus)  is  the  most  important.  The  organism  is  present  in  variable 
but  small  numbers  in  the  feces  of  healthy  adults,  and  occasionally  in 
young  children  as  well.  It  may  occasionally  become  a  very  prominent 
organism  among  the  fecal  flora.  The  isolation  and  recognition  of  the 
gas  bacillus  from  the  intestinal  contents  depends  primarily  upon  the 
energetic  fermentation  in  milk  cultures  inoculated  with  feces  and 
heated  to  80°  C.  for  twenty  minutes  prior  to  incubation.  (See  Chapter 
XXV  for  details.)  Members  of  the  spiral  group,  including  the  highly 
pathogenic  cholera  vibrio,  are  readily  isolated  and  identified  by  the 
procedure  described  in  the  section  on  Vibrio  Choleras  (Chapter  XXVI). 

Tubercle  bacilli  are  not  infrequently  found  in  the  feces  of  individuals 
who  have  advanced  pulmonary  tuberculosis.  It  is  almost  certain 
that  the  organisms  have  been  swallowed  in  a  majority  of  such  cases. 
Occasionally  a  diagnosis  of  tuberculosis  may  be  made  thus  in  young 
children  from  whom  it  is  difficult  or  impossible  to  obtain  a  satisfactory 
specimen  of  sputum.  Tubercle  bacilli  are  also  found  in  the  feces, 
derived  from  tuberculous  ulcerations.  A  diagnosis  of  tubercle  bacillus 
cannot  safely  be  made  from  a  demonstration  of  acid-fast  organisms  in 
the  fecal  contents,  because  acid-fast  bacteria  other  than  tubercle 
bacilli  may  be  present.  A  guinea-pig  furnishes  the  only  reliable 
method  of  distinguishing  tubercle  bacilli  from  adventitious  non-patho- 
genic acid-fast  organisms. 

Examination  of  Sputum,  of  Buccal  and  Pharyngeal  Material.1— 
A  sample  of  sputum  suitable  for  bacteriological  examination  should  be 
collected  with  care.  The  mouth  should  be  clean,  the  receptacle  should 
be  sterile,  and  the  material  should  be  raised  by  a  deep  pulmonary 
cough,  not  by  a  superficial  effort.  Buccal  and  pharyngeal  material 
for  bacteriological  examination  is  usually  obtained  upon  sterile  cotton 
swabs.  Bits  of  membrane  may  be  removed  with  sterile  forceps. 

Examination  by  Staining. — A  Gram-stained  preparation  of  sputum, 
buccal  or  pharyngeal  material  usually  contains  a  variety  of  micro- 
organisms comprising  cocci,  spiral  forms,  and  even  fungi  and  yeasts. 
Many  of  the  organisms  may  be  normal  inhabitants  of  the  buccal 

1  An  excellent  discussion  of  Infections  of  the  Respiratory  Tract  and  of  Sputum  as  a 
Moans  of  Diagnosis  is  that  of  Leutscher,  Arch.  Int.  Med.,  1915,  xvi,  657. 


MATERIAL  FROM  THE  LIVING  SUBJECT  233 

cavity,  and  of  the  pathogenic  organisms,  pneumococci,  streptococci, 
and  occasionally  diphtheria  bacilli  are  found.  Usually  clinical  signs 
or  an  abnormal  appearance  of  the  sputum,  mouth,  or  throat  lead 
to  a  microscopic  examination  of  the  material  from  this  region  and,  as 
a  rule,  the  nature  of  the  symptomatology  is  a  reliable  guide  to  the 
stain  to  be  used.  Among  the  organisms  which  stain  by  Gram's  method, 
pneumococci,  streptococci,  staphylococci,  Micrococcus  tetragenus, 
and  occasionally  Diplococcus  crassus  are  the  more  common  spherical 
organisms.  Micrococcus  catarrhalis,  the  meningococcus  and  para- 
mcningococcus  are  the  only  Gram-negative  cocci,  so  far  as  is  known. 

Of  the  Gram-staining  bacilli,  the  diphtheria  and  pseudodiphtheria 
bacilli  together  with  Bacillus  subtilis  and  rarely  Bacillus  anthracis 
may  be  found.  The  bacillus  of  Friedlander,  typhoid,  influenza,  pertus- 
sis, plague  and  glanders  bacilli  are  Gram-negative,  Bacillus  fusiformis 
and  Vincent's  spirillum  are  Gram-negative  as  well.  They  color  some- 
what indistinctly  with  Lofflers  methylene  blue  and  very  distinctly 
with  Wright's  or  Giemsa's  stain.  Mouth  spirals  and  Treponema 
pallidum  are  best  stained  with  the  latter  method.  Tubercle,  leprosy 
and  nasal  secretion  bacilli  (Karlinski)  stain  with  the  acid-fast  stain. 

Higher  bacteria  and  moulds  are  occasionally  identified  in  material 
from  the  buccal  cavity.  Actinomyces,  Oi'dium  albicans,  aspergillus, 
mucor,  streptothrix,  and  yeasts  have  been  detected.  The  virus  of 
poliomyelitis  has  also  been  demonstrated  in  material  from  the  naso- 
pharynx which  has  been  freed  from  bacteria  by  passage  through  a 
Berkefeld  filter  and  injected  into  a  monkey. 

For  the  routine  examination  of  sputum,  three  stains  are  ordinarily 
employed — Ziehl-Neelsen  for  tubercle  bacilli,  Loffler's  alkaline  methyl- 
ene blue  for  diphtheria,  pseudodiphtheria,  and  fusiform  bacilli  (and 
Vincent's  spirillum),  and  the  Gram  stain,  using  dilute  carbol  fuchsin 
as  a  counterstain  for  pneumococci,  streptococci,  influenza,  and  per- 
tussis bacilli  principally.  Smith's  stain  for  sputum  (see  page  186) 
is  advantageous  for  pneumonic  sputum. 

The  organisms  mentioned  previously  but  not  detailed  in  the  routine 
examination  of  sputum  are  of  comparatively  rare  occurrence.  They 
must  be  studied  by  purely  cultural  methods. 

Cultural  Methods. — Antiseptic  gargles  should  not  be  used  before 
collecting  sputum  or  material  from  the  mouth  or  pharynx  for  cultural 
examination.  Sputum  or  exudate,  obtained  in  a  suitable  manner,  is 
first  washed  through  six  or  seven  portions  of  sterile  salt  solution,  if  its 
cohesiveness  permits,  to  remove  or  diminish  surface  contamination. 


234         BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL 

For  a  majority  of  bacteria,  freshly  prepared  blood  agar  plates  are  the 
most  satisfactory  media  to  employ.1  Hemolytic  streptococci,  pneumo- 
cocci,  Pneumococcus  mucosus  and  influenza  bacilli  grow  upon  this 
medium. 

Diphtheria  bacilli  are  grown  upon  Loffler's  blood  serum,  as  described 
in  the  section  on  diphtheria. 

Tubercle  bacilli  can  be  readily  distinguished  from  lepra  bacilli, 
nasal  secretion  bacilli  and  adventitious  acid-fast  organisms  by  the 
injection  of  washed,  cheesy  particles  from  sputum  into  guinea-pigs. 

The  organism  commonly  found  in  Vincent's  angina  (Bacillus  fusi- 
formis)  is  not  readily  cultivated  upon  ordinary  media.  Its  recognition 
usually  depends  upon  its  demonstration  in  smears  prepared  directly 
from  the  lesions. 

Bacteriological  Examination  of  the  Eye. — The  normal  conjunctival 
sac  frequently  contains  Staphylococcus  albus  and  Bacillus  xerosis; 
indeed  these  organisms  are  so  commonly  found  in  this  region  that  they 
are  regarded  as  normal  inhabitants.  Abnormally  a  variety  of  bacteria 
may  develop  on  the  conjunctiva,  frequently  causing  a  violent  inflam- 
mation. Material  for  bacteriological  examination  is  best  obtained 
after  gently  flooding  the  conjunctival  sac  with  a  few  drops  of  sterile 
salt  solution,  which  are  removed  with  a  sterile  cotton  swab.  Then  a 
small  sterile  cotton  swab  is  gently  rubbed  over  the  conjunctival  sur- 
face and  inoculated  into  suitable  media  after  a  Gram-stained  smear 
has  been  examined. 

The  gonococcus,  Koch- Weeks  bacillus,  and  the  pneumococcus  are 
more  commonly  the  incitants  of  acute  inflammation  of  the  conjunctiva; 
less  frequently  hemoglobinophilic  bacilli  (B.  influenzse  particularly) 
or  Bacillus  pyocyaneus  may  be  found.  An  examination  of  Gram- 
stained  smears  will  indicate  the  media  to  be  employed  if  isolation  of 
the  organisms  in  pure  culture  is  desired.  The  meningococcus  is  occa- 
sionally found  in  conjunctival  inflammations  in  cases  of  cerebrospinal 
meningitis;  it  must  not  be  confused  with  the  gonococcus.  Micrococcus 
catarrhalis,  which  resembles  both  the  gonococcus  and  meningococcus 
in  its  morphology  and  staining  reactions,  does  not  produce  an  acute 
conjunctival  inflammation  with  a  profuse  purulent  discharge — rather, 
this  organism  usually  gives  rise  to  a  slight  reaction,  even  though  the 


1  Several  drops  of  sterile  blood,  obtained  from  the  finger  or  the  lobe  of  the  ear  after 
a  preliminary  sterilization,  are  placed  in  the  centre  of  an  agar  plate.  The  material  to 
be  studied  is  streaked  out  radially  from  the  blood.  Enough  blood  can  be  moved  with 
the  organisms  by  this  method  to  insure  growth. 


MATERIAL  FROM  THE  LIVING  SUBJECT  235 

organisms  are  numerous.1  Blood  agar  plates  are  preferable  for  the 
cultivation  of  bacteria  from  the  eye.  Not  only  do  the  hemoglo- 
binophilic  organisms  and  the  gonococcus  grow  in  this  medium — the 
less  fastidious  forms  also  develop  rapidly. 

Subacute  Conjunctivitis. — The  Morax-Axenfeld  bacillus  is  a  common 
excitant  of  subacute  conjunctivitis,  particularly  when  the  internal 
angle  is  involved.  The  secretior  is  meagre  and  best  obtained  in  the 
morning.  The  bacilli  are  short  aud  thick,  Gram  negative,  and  occur 
singly  and  in  pairs,  both  free  and  in  leukocytes.  They  must  be  dis- 
tinguished from  members  of  the  Mucosus  Capsulatus  Group,  which  are 
comparatively  common  in  ozena  which  involves  the  nasal  ducts.  The 
latter  are  capsulated,  which  distinguishes  them  from  the  Morax-Axen- 
feld organism. 

Corneal  ulcerations  may  be  caused  by  pneumococci,  streptococci, 
leprosy  bacilli,  and  rarely  by  tubercle  bacilli.  The  latter  organism 
is  best  detected  by  animal  inoculation. 

Pseudomembranous  conjunctivitis  is  frequently  the  result  of  a 
localization  and  development  of  diphtheria  bacilli,  less  commonly  of 
streptococci.  The  etiology  of  phlyctenular  conjunctivitis  is  still 
unknown. 

Bacteriological  Examination  of  the  Ear  and  Nose. — The  middle 
ear  normally  is  sterile,  but  bacteria  may  reach  it  either  by  extension 
of  growth  from  the  nasopharynx  through  the  Eustachian  tube,  or 
directly  from  the  blood  and  lymph  channels.  By  far  the  most  com- 
mon incitant  of  infection  of  the  middle  ear  is  the  streptococcus  alone 
or  less  frequently  in  association  with  other  organisms.  This  organism 
is  also  commonly  isolated  from  thrombosed  sinuses.  The  pneumococcus 
and  Pneumococcus  mucosus  are  also  frequently  isolated  from  otitis 
media.  Bacillus  pyocyaneus  or  Bacillus  proteus  are  not  uncommonly 
found  in  middle  ear  infections,  particularly  those  containing  fetid  pus. 
Bacillus  coli  has  also  been  detected  in  foul-smelling  pus  from  the 
middle  ear.  Staphylococci,  Micrococcus  catarrhalis,  Micrococcus 
tetragenus,  influenza  bacilli,  members  of  the  Mucosus  Capsulatus 
Group  of  bacilli,  typhoid  and  diphtheria  bacilli  have  also  been  isolated 
from  otitis  media. 

Infection  of  the  external  auditory  meatus,  which  contains  cerumen, 
is  frequently  the  result  of  an  overgrowth  of  various  moulds,  particularly 
Aspergillus  and  Mucor. 

1  For  a  discussion  of  Gram-negative  diplococci  found  in  the  eye,  see  Blue,  Arch. 
Ophthal.,  1915,  xliv,  No.  6. 


236         BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL 

The  normal  nasal  cavity,  although  freely  exposed  to  the  exterior 
and  theoretically,  at  least,  continually  contaminated  with  bacteria 
both  from  the  inspired  air  and  the  microorganisms  washed  from  the 
eyes  in  the  lachrymal  secretions,  is  relatively  free  from  microorganisms. 
Staphylococcus  albus,  non-hemolytic  short-chain  streptococci  and 
pseudodiphtheria  bacilli  appear  to  be  the  more  common  organisms 
isolated  from  the  healthy  nasal  cavity.  Material  for  examination  is 
obtained  after  cleaning  the  external  nares  with  sterile  salt  solution 
upon  swabs  of  sterile  cotton. 

Diphtheria,  leprosy,  ozena,  rhinoscleroma  and  various  coryzas  are 
the  common  types  of  nasal  infection,  but  a  variety  of  organisms  may 
be  present  there  either  transiently,  or  somewhat  more  permanently 
during  bronchial  infections.  Thus  pneumococci,  influenza  and  per- 
tussis bacilli  have  occasionally  been  isolated  from  the  nasal  secretion 
during  pneumonia,  influenza  or  whooping  cough  respectively.  Menin- 
gococci  and  parameningococci  have  been  demonstrated  both  in  patients 
and  carriers  during  epidemics  of  cerebrospinal  meningitis.  It  is  not 
unlikely  that  Micrococcus  catarrhalis  has  been  incorrectly  diagnosed 
as  the  meningococcus  in  the  past,  because  both  organisms  are  Gram- 
negative  diplococci.  Microcococcus  catarrhalis  is  occasionally  found 
in  large  numbers  in  the  nasal  secretion  of  acute  coryza. 

The  bacteriology  of  ozena  is  a  subject  of  controversy.  Bacillus 
ozaense  and  Bacillus  rhinoscleromatis,  both  members  of  the  Mucosus 
Capsulatus  Group  of  bacteria,  have  been  regarded  as  the  etiological 
agents  in  the  past. 

The  earliest  lesion  of  leprosy  appears  to  be  a  nasal  ulcer,  more 
frequently  located  at  the  junction  of  the  bony  and  cartilaginous  septum, 
hence  an  examination  of  the  nasal  cavity  is  of  paramount  importance 
for  the  early  diagnosis  of  this  disease. 

Tuberculous  ulcerations  of  the  nose  are  comparatively  infrequent; 
the  tubercle  bacillus  is  readily  distinguished  from  the  lepra  bacillus 
by  injection  of  suspected  material  into  a  guinea-pig.  The  animal 
is  very  susceptible  to  infection  with  the  tubercle  bacillus,  but  refrac- 
tory to  lepra  bacilli.  Occasionally  acid-fast  bacilli,  which  are  neither 
lepra  nor  tubercle  bacilli,  have  been  reported  as  occurring  in  the 
nasal  secretion.  Karlinski's  nasal  secretion  bacillus  is  the  best  known 
of  the  Saprophytic  Acid-fast  Group.  It  grows  promptly  and  with  con- 
siderable luxuriance  upon  glycerin  agar,  which  at  once  distinguishes 
it  from  the  pathogenic  acid-fast  bacilli. 

Nasal  diphtheria  is  not  an  uncommon  type  of  infection  with  the 


UTILIZATION  OF  ANIMALS  FOR  BACTERIAL  DIAGNOSIS      237 

diphtheria  bacillus.  The  organism  is  readily  distinguished  by  its 
morphology  with  the  methylene  blue  stain  both  from  the  nasal  secre- 
tion and  from  cultures  upon  Loffler's  blood  serum.  When  the  nasal 
secretion  is  profuse,  as,  for  example,  in  acute  or  subacute  coryza, 
saprophytic  bacteria,  as  Bacillus  proteus,  may  develop  in  the  nasal 
secretion,  causing  extremely  offensive  odors.  There  is  little  evidence 
that  the  organism  is  exciting  inflammation,  however;  it  would  appear 
that  the  secretion  is  p  favorable  medium  for  the  development  of  the 
organism. 

The  virus  of  poliomyelitis  may  be  found  in  the  nasal  secretion.  Its 
identification  has  been  discussed  above. 

THE  UTILIZATION  OF  ANIMALS  FOR  BACTERIAL  DIAGNOSIS 
AND   EXPERIMENTATION. 

Pasteur's  brilliant  animal  experiments  led  Koch  to  formulate*  his 
Postulates  for  the  etiological  relationship  of  bacteria  to  disease.  A 
rigorous  demonstration  of  the  etiological  relationship  of  bacteria  to 
specific  disease,  said  Koch,  must  fulfill  the  following  conditions: 

1.  A  specific  microorganism  must  be  constantly  associated  with 
the  disease. 

2.  The  organism  must  be  isolated  from  the  lesion  and  cultivated 
outside  the  body  of  the  host. 

3.  A  pure  culture  of  the  organism  must  incite  the  disease  when 
introduced  into  a  normal  animal. 

4.  The  organism  must  be  isolated  from  the  experimental  animal 
again  in  pure  culture. 

Experience  has  shown  that  many  diseases  of  man  cannot  be  exactly 
reproduced  in  experimental  animals  and  Koch's  Postulates,  therefore, 
cannot  be  fulfilled  with  exactitude  in  these  instances.  Nevertheless, 
experimental  animals  are  indispensable  both  in  diagnostic  and 
experimental  bacteriological  laboratories.  They  are  used: 

1.  As  culture  media  for  certain  types  of  bacteria  which  grow  slowly 
or  feebly  upon  artificial  media,  particularly  when  the  number  of  such 
organisms  is  too  small  to  permit  of  cultivation  under  artificial  condi- 
tions.   The  isolation  of  tubercle  bacilli  from  urine,  of  glanders  bacilli 
from  the  lesions  of  glanders  are  illustrative. 

2.  To  obtain  pure  cultures  of  bacteria  from  mixtures,  as  the  inocu- 
lation of  white  mice  with  pneumonic  sputum  for  the  pneumococcus, 
or  rubbing  mixtures  containing  plague  bacilli  upon  the  shaved  abdo- 
men of  a  guinea-pig  to  obtain  pure  cultures  of  B.  pestis. 


238         BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL 


3.  To  study  experimentally  the   lesions  incited  by  specific  micro- 
organisms. 

4.  To  distinguish  sharply  between  closely  related  bacteria,  as  for 
example,  between  bovine  and  human  tubercle  bacilli.    Thus,  rabbits 


FIG.  31. — Guinea-pig  dissection  to  show  anatomical  relations  of  internal  organs  and 
important  lymph  glands.    (From  Eyre,  Bacteriological  Technique,  Saunders  &  Co.) 

are  susceptible  to  infection  with  bovine,  but  not  with  human  tubercle 
bacilli.    Guinea-pigs  are  susceptible  to  infection  with  both  types. 

5.  To  study  the  virulence  of  various  microorganisms. 

6.  To  test  the  toxicity  of  bacterial  toxins  and  other  products,  and 
to  measure  the  potency  of  curative  sera, 


UTILIZATION  OF  ANIMALS  FOR  BACTERIAL  DIAGNOSIS      239.^ 

7.  For  the  production  of  various  antibodies,  as  antitoxins,  agglu- 
tinins,  precipitins  and  lysins. 

The  choice  of  animals  depends  chiefly  upon  the  nature  of  the  obser- 
vation to  be  made.  Rabbits,  guinea-pigs,  white  rats  and  mice,  dogs 
and  cats  are  more  commonly  made  use  of  for  these  various  examina- 
tions. The  method  and  site  of  inoculation,  as  well  as  the  dosage,  may 
influence  the  course  of  the  infection. 

The  Inoculation  of  Animals. — Animals  may  be  inoculated  through 
natural  channels,  as  by  inhalation  into  the  respiratory  tract,  or  inges- 
tion  into  the  alimentary  tract.  More  frequently,  however,  material  is 
introduced  parenterally  into  the  tissues  direct.  The  site  of  inoculation 
is  usually  the  skin,  the  body  fluids  or  body  cavities.  The  skin  must 
necessarily  be  entered  to  reach  the  deeper  tissues.  For  this  reason 
the  site  of  injection  should  be  shaved  and  sterilized  with  tincture  of 
iodin.1 

Cutaneous  Inoculation. — (a)  Cutaneous:  Material  is  rubbed  upon  a 
shaved  area  of  skin. 

(6)  Intracutaneous :    Injection  is  made  directly  into  the  skin. 

(c)  Subcutaneous:  Material  is  introduced  beneath  the  skin.  A 
pocket  is  sometimes  made  by  separating  the  skin  from  the  cellular 
subcutaneous  tissue,  into  which  solid  fragments  of  tissue  are  placed. 
The  skin  over  the  abdomen  is  a  common  site  for  inoculation  with  fluid 
cultures;  the  hypodermic  needle  is  introduced  at  one  side  of  the 
median  line  and  forced  through  the  subcutaneous  tissue  in  a  trans- 
verse direction,  to  a  point  well  beyond  the  median  line  on  the  opposite 
side.  The  abdominal  wall  becomes  somewhat  tense  and  does  not 
permit  leakage  to  the  outside  if  this  procedure  is  followed. 

Intravenous  inoculations  are  made  either  into  the  blood  stream 
through  a  vein,  or  directly  into  the  heart.  Rabbits  are  readily  injected 
through  the  marginal  ear  veins;  the  vein  is  pinched  close  to  the  head 
of  the  animal  and  gently  massaged;  this  causes  distention  and  makes 
the  vein  prominent.  A  hypodermic  needle  will  then  readily  enter 
the  vein;  it  should  be  gently  forced  along  its  course  for  a  centimeter 
or  two  before  injection. 

Body  Cavities. — The  peritoneal  cavity  is  commonly  selected,  but 
intrapleural  injections  are  readily  made.  Before  introducing  a  hypo- 
dermic needle  into  the  peritoneal  cavity,  the  animal,  guinea-pig  or 
» 

1  Tincture  of  iodin  should  be  freshly  prepared  and  painted  upon  the  dry  surface  it  is 
desired  to  sterilize.  Sterilization  is  usually  accomplished  after  two  or  three  minute's 
exposure  to  the  iodin  solution. 


240         BACTERIOLOGICAL  EXAMINATION  OF  MATERIAL 

rabbit,  is  held  head  downward  to  permit  the  intestines  to  pass  ante- 
riorly as  far  as  possible.  The  needle  is  first  introduced  somewhat 
obliquely  through  the  abdominal  skin  posteriorly,  then  directly  into 
a  fold  of  the  abdominal  wall  pinched  between  the  fingers.  The  needle 
should  be  pressed  in  until  resistance  to  its  passage  has  ceased.  Unless 
the  precaution  is  taken  to  dip  the  point  of  the  needle  in  sterile  vase- 
line, some  of  the  contents  will  be  introduced  into  the  cutaneous  or 
subcutaneous  tissues  as  well  as  the  peritoneal  cavity.  The  u  Plitchens" 
syringe  with  its  side-arm  containing  salt  solution  to  rinse  the  entire 
charge  from  the  needle  before  withdrawal  from  the  animal  is  highly 
recommended  for  this  purpose. 

Intracerebral  injections  are  made  either  through  the  optic  foramen, 
or  through  the  dura  after  trephining  the  skull. 

Intratracheal  injections  are  occasionally  made,  but  more  commonly 
the  material  is  introduced  deep  into  the  bronchi  through  a  flexible 
rubber  cannula.  The  animal  should  be  anesthetized  for  this  operation. 

White  mice  and  rats  are  usually  inoculated  in  the  loose  subcutaneous 
tissue  at  the  base  of  the  tail.  The  needle  should  pass  somewhat 
obliquely  to  avoid  the  spinal  cord. 

Care  of  Animals. — Guinea-pigs  and  rabbits  are  very  susceptible  to 
"snuffles"  and  frequently  perish  from  contagious  pneumonia  and 
other  epizootics  of  the  respiratory  tract.1  The  first  symptoms  are 
usually  nasal  discharge  and  a  mucopurulent  exudation  from  the  eyes. 
Such  animals  should  be  killed  at  once  and  their  cages  thoroughly 
sterilized.  Animals  in  adjacent  cages  should  be  quarantined. 

Inoculated  animals  are  best  kept  in  separate  cages  apart  from  the 
healthy  stock.  If  they  become  moribund  it  is  better  to  chloroform 
them  and  perform  the  autopsy  at  once;  fresh,  uncontaminated  cul- 
tures may  be  obtained  only  at  this  time.  If  animals  are  permitted 
to  die,  frequently  several  hours  intervene  before  an  autopsy  is  per- 
formed, and  postmortem  bacterial  invasion  of  the  tissues  and  blood 
stream  is  usually  a  disturbing  factor.  Infected  material  is  obtained 
from  animals  with  the  same  precautions  and  technic  as  those  for  a 
human  autopsy. 

1  Theobald  Smith,  Jour.  Med.  Research,  xxix,  291,  for  discussion. 


CHAPTER  XL 


PRACTICAL  STERILIZATION,  ANTISEPSIS  AND 
DISINFECTION. 


LABORATORY  STERILIZATION. 
Physical  Agents. 

Heat. 

Live  Steam. 

Fractional  Sterilization. 

Boiling  Water. 
Chemical  Solutions. 

Sajts  of  Heavy  Metals. 

Oxidizing  Solutions. 

Phenols,  Cresols. 

Tincture  of  lodin. 

Boric  Acid. 

Formaldehyde. 

Essential  Oils. 

Soaps. 
Testing    and    Standardizing 

Disinfectants. 


Liquid 


Gaseous  Disinfectants. 
Formaldehyde . 
Paraform. 
Sulphur. 
Chlorine  Gas. 
Ozone. 
PRACTICAL  DISINFECTION. 

Sputum: 

Vomitus. 

Feces  and  Urine. 

Fomites. 

Bath  Water. 

Skin  and  Hand. 

Instruments. 

Clinical  Thermometers — Dental  Instru- 
ments. 


THE  terms  sterilization,  disinfection,  antisepsis  and  deodorization 
are  frequently  used  indiscriminately,  but  it  is  important  to  distinguish 
between  them.  Sterilization  and  disinfection  imply  the  destruction 
of  microorganisms,  the  latter  being  restricted  largely  to  hygienic 
procedure,  as  the  disinfection  of  excreta,  etc.  A  restriction  of  bac- 
terial growth  not  necessarily  involving  the  death  of  microorganisms 
'is  properly  termed  antisepsis.  Deodorants,  as  the  term  signifies,  are 
those  substances  which  destroy  or  mask  odors;  deodorants  may  or 
may  not  destroy  bacteria. 


LABORATORY    STERILIZATION. 

The  many  kinds  of  apparatus  and  media  used  in  the  study  of  bac- 
teria must  be  freed  from  adventitious  organisms  before  they  are 
applicable  to  bacteriological  investigation.  Physical  and  chemical 
agents  are  commonly  made  use  of  for  this  purpose. 

Physical  Agents. — 1.  Heat. — (a)  Incineration. — Incineration  is  a 
most  efficient  method  of  sterilizing  articles  of  little  value.  The  free 
flame  is  commonly  used  for  sterilizing  platinum  needles  and  platinum 
loops.  If  the  latter  are  charged  with  pathogenic  bacteria,  and  par- 
ticularly bacteria  which  contain  fats,  as  the  tubercle  bacillus,  it  is 

% 

16 


242          STERILIZATION,  ANTISEPSIS  AND  DISINFECTION 

necessary  to  dry  the  material  by  holding  the  loop  near  the  flame  before 
incineration  to  prevent  "spattering."  The  "bacteria  incinerator" 
made  by  de  Khotinsky  is  particularly  to  be  recommended  for  this 
purpose.1 

(6)  Dry  Heat. — Test  tubes,  flasks,  Petri  dishes,  pipettes  and  other 
laboratory  glassware  are  sterilized  in  the  hot-air  sterilizer — an  oven 
heated  with  a  gas  flame.  An  exposure  of  one  and  a  half  hours  at  160° 
C.  or  one  hour  at  180°  C.  will  effectually  kill  all  spores.  The  heat 
should  be  applied  gradually  and  reduced  gradually  to  diminish  the 
danger  of  cracking.  Dry  heat  has  but  little  power  of  penetration. 
Glassware  is  conveniently  wrapped  in  paper  before  sterilization  to 
protect  it  from  dust  prior  to  its  use.  The  cotton  plugs  of  flasks  and 
beakers  are  also  covered  with  paper  before  sterilization,  for  the  same 
reason. 

(c)  Moist  Heat. — 1.  The  most  satisfactory  agent  for  the  steriliza- 
tion of  articles  uninjured  by  moisture  is  steam  under  pressure.  Many 
kinds  of  media  and  laboratory  apparatus,  and  fomites  as  well  are 
quickly  and  completely  sterilized  by  steam.  The  autoclave  is  com- 
monly used  for  laboratory  purposes.  It  consists  essentially  of  a 
double-walled  chamber  with  close-fitting  cover,  into  which  steam 
may  be  introduced.  There  are  many  patterns,  but  the  essential 
features  are — the  steam  should  enter  the  chamber  from  the  top,  and 
the  bottom  of  the  chamber  should  be  provided  with  a  stop-cock, 
through  which  the  residual  air  and  condensation  can  escape. 

Operation. — A  single  layer  of  apparatus  should  be  sterilized  at  one 
time.  If  several  layers  are  introduced,  condensation  water  from  the 
upper  layer  may  collect  on  the  lower  layers,  permitting  of  subsequent 
contamination.  Steam  is  admitted  to  the  chamber  to  displace  the 
air,  and  the  air-cock  should  remain  open  until  live  steam  flows  freely 
from  it,  because  hot  air  is  far  less  efficient  than  steam  for  sterilization. 
Also,  the  condensed  steam  escapes  through  the  same  orifice.  When 
all  the  air  is  replaced  by  dry  steam  the  pressure  is  gradually  in- 
creased until  fifteen  pounds  are  recorded  on  the  pressure  gauge. 
This  pressure  is  maintained  from  ten  to  twenty  minutes,  depending 
upon  the  nature  of  the  material  to  be  sterilized.  In  general,  media 

1  It  consists  essentially  of  a  tube  about  12  cm.  in  length  and  1  cm.  in  diameter,  of 
fire  clay  surrounded  by  a  resistance  coil  of  sufficiently  fine  wire  and  numerous  layers 
to  heat  the  interior  of  the  tube  to  a  white  heat.  The  charged  platinum  wire  is  placed 
in  the  tube,  and  within  a  few  seconds  it  becomes  white-hot.  There  is  absolutely  no  danger 
from  "spattering,"  because  the  extruded  organisms  fall  upon  the  hot  walls  of  the  tube 
(see  Fig.  23,  page  205). 


LABORATORY  STERILIZATION  243 

in  test-tubes  is  more  quickly  sterilized  than  media  in  flasks.  At  the 
end  of  the  allotted  time,  the  pressure  is  gradually  reduced  until 
equilibrium  is  reached  with  the  atmospheric  pressure;  a  sudden  release 
of  pressure  would  cause  violent  ebullition  of  fluid,  and  a  wetting  or 
even  expulsion  of  cotton  plugs  from  test-tubes  or  flasks. 

TABLE   OF   PRESSURE   AND   TEMPERATURE. 

Pressure,  Temperature, 

pounds.  Centigrade. 

0  100.0° 

5  107.7° 

10  115.5° 

15  121.5° 

20  126.5° 

2.  Live  Steam. — Many  solutions  are  injured  by  temperatures  above 
100°  C.    Media  containing  sugars  (particularly  bioses)  milk  and  gela- 
tin are  partly  decomposed  by  prolonged  sterilization  in  the  autoclave. 
An  exposure  to  live  steam  at  lOQ^.C.  for  thirty  minutes  on  each  of 
three  successive  days  usually  suffices  to  effect  sterilization  of  these 
media  without  injury  to  the  constituents  of  the  medium.    This  method 
of  fractional  sterilization  depends  upon  the  destruction  of  all  vegetative 
cells  during  the  heating  process,  and  the  germination  of  spores  into 
vegetative  organisms  between  heatings.    It  is  assumed  that  all  viable 
spores  will  have  germinated  before  the  third  exposure  to  heat,  but 
Theobald  Smith1  has  shown  that  spores  of  anaerobic  bacteria  may  not 
vegetate  within  the  specified  time.    A  fourth  exposure  to  heat  after 
two  or  three  days  may  be  required  to  insure  sterilization.    The  Arnold 
sterilizer  is  widely  used  for  fractional  sterilization  with  live  steam. 
It  consists  essentially  of  a  double-walled  copper  chamber  surmounting 
a  double-bottomed  water  reservoir,  the  lower  compartment  of  which 
is  shallow  and  contains  but  little  water.     A  flame  applied  to  this 
shallow  reservoir  soon  generates  steam,  which  rises  through  a  central 
passage  to  the  chamber  in  which  the  material  to  be  sterilized  is  placed. 
Condensed  steam  flows  by  gravity  to  the  upper  water  compartment, 
and  from  thence  to  the  lower  heated  reservoir  to  replace  the  evapora- 
tion.   It  takes  but  a  few  minutes  to  generate  sufficient  steam  to  fill 
the  sterilizing  chamber.     The  sterilizing  process  begins  when  the 
contents  of  the  sterilizing  chamber  have  reached  100°  C. 

3.  Fractional  Sterilization  at  temperatures  from  60°  to  80°  C.  is  fre- 
quently made  use  of  for  materials  such  as  blood  serum,  which  would 
be  injured  by  exposure  to  100°  C.    The  sterilizing  process  is  repeated 

1  Jour.  Exp.  Med.,  1898,  iii,  647. 


244          STERILIZATION,  ANTISEPSIS  AND  DISINFECTION 

for  an  hour  daily  over  a  period  of  five  to  seven  days.  The  sterilization 
of  Loffler's  blood  serum  in  a  Koch  inspissator  is  carried  out  at  this 
lower  temperature. 

4.  Boiling  Water. — Petri  dishes,  culture  tubes  and  other  apparatus 
containing  pathogenic  bacteria  may  be  freed  from  bacteria  by  boiling 
in  water  for  five  minutes.  Practically  no  pathogenic  bacteria  form 
spores.  If  tetanus,  anthrax  or  gas  bacillus  cultures  are  to  be  destroyed, 
the  autoclave  is  necessary. 

Chemical  Solutions. — Chemical  disinfectants  are  most  efficient  in 
aqueous  solutions,  and  they  must  therefore  be  soluble  in  water. 
Moisture  is  also  essential  for  gaseous  disinfectants. 

The  theory  of  the  germicidal  action  of  disinfectants  is  not  well 
understood;  apparently  the  efficiency  of  salts  of  heavy  metals  is 
associated  with  their  noteworthy  affinity  for  proteins,  with  which  they 
form  firm  combinations.  It  must  be  remembered  that  these  salts 
react  more  quickly  with  animal  proteins  than  bacterial  proteins, 
therefore  greater  concentrations  of  metallic  salts  are  required  to  kill 
bacteria  suspended  in  protein  solutions  than  to  destroy  the  same 
organisms  in  aqueous  suspension.  Thus,  typhoid  bacilli  may  be 
killed  by  1  to  500,000  bichloride  of  mercury  if  they  are  suspended  in 
water,  but  a  concentration  of  at  least  1  to  1500  is  required  to  sterilize 
the  same  organism  in  blood  serum.  Absolute  alcohol  does  not 
appear  to  be  a  very  powerful  germicide;  possibly  its  rather  limited 
germicidal  value  is  associated  with  its  dehydrating  properties.  Dilute 
solutions  of  alcohol,  20  to  30  per  cent.,  are  practically  as  destructive 
of  bacteria  as  absolute  alcohol  is.  Phenols  are  excellent  germicides 
in  aqueous  solutions,  but  their  tendency  to  go  into  solution  in  oils 
(which  do  not  readily  penetrate  the  ectoplasm  of  cellular  structures) 
makes  them  unreliable  germicides  in  oily  menstrua. 

Salts  of  Heavy  Metals. — 1.  Mercuric  Chloride,  HgCI2. — Mercuric 
chloride  or  bichloride  of  mercury  is  a  powerful  germicide,  very  soluble 
in  hot  water,  less  soluble  in  cold  water.1  It  is  usually  dispensed  in 
tablet  form  mixed  with  NaCl,  which  increases  its  solubility  and  also 
prevents  somewhat  its  marked  tendency  to  unite  with  proteins.  This 
is  of  importance  in  the  treatment  of  wounds  and  secretions  of  wounds 
with  this  germicide.  A  1  to  1000  solution  of  bichloride  in  water  is 
the  dilution  commonly  used  for  practical  purposes.  This  strength 

1  One  part  of  bichloride  will  dissolve  in  3  to  4  parts  of  boiling,  distilled  water;  upon 
cooling,  much  of  the  bichloride  becomes  insoluble;  one  part  of  the  salt  will  dissolve  in 
16  to  18  parts  of  water  at  room  temperature. 


LABORATORY  STERILIZATION  245 

of  solution  will  kill  all  pathogenic  bacteria  in  a  very  short  time;  a 
solution  of  1  to  500  strength  will  even  kill  anthrax  spores  within  a 
few  hours. 

The  advantage  of  bichloride  of  mercury  as  a  germicide  resides  in 
its  great  bactericidal  powers.  Its  disadvantages  are:  its  marked 
affinity  for  protein  which,  in  the  case  of  wounds,  may  lead  to  local 
necrosis  of  tissue,  or  in  greater  concentrations,  by  absorption,  to 
toxic  action  on  the  kidneys,  intestinal  tract,  and  even  the  central 
nervous  system.  It  is  unreliable  for  the  disinfection  of  sputum,  feces, 
urine,  purulent  discharges,  and  other  excreta,  and  it  should  never  be 
employed  in  the  sterilization  of  instruments  or  eating  utensils.  Linen 
soiled  with  blood  or  stained  in  any  way  should  not  be  immersed  in 
bichloride,  for  it  acts  as  a  mordant  and  "sets"  the  stain. 

2.  Silver  Salts. — Silver  nitrate  is  a  much  less  efficient  germicide 
than  mercuric  chloride,  but  it  is  quite  extensively  used  upon  mucous 
membranes.  The  soluble  organic  compounds  of  silver,  as  Protargol, 
are  less  irritating  than  the  inorganic  salts  and  apparently  nearly  as 
efficient. 

Oxidizing  Solutions. — 1.  Potassium  Permanganate,  KMn04. — Potas- 
sium permanganate  is  a  strong  disinfecting  agent,  but  it  is  almost 
instantly  reduced  and  rendered  inert  by  organic  substances.  This 
greatly  impairs  its  practical  value.  Nevertheless,  it  is  used  in  surgical 
asepsis  and  also  in  wells  and  cisterns  which  are  to  be  freed  from 
pathogenic  bacteria.  A  strong  solution  is  thrown  into  the  well  or 
cistern,  enough  to  impart  a  very  pronounced  pink  color  to  the  water, 
and  left  for  several  hours.  The  water  is  fit  for  use  when  the  last  traces 
of  color  are  removed  by  dilution  or  emptying  and  washing  out  the 
reservoir.  This  process  is  spoken  of  as  "pinking"  a  well. 

2.  Hydrogen  Peroxide,   H2O2 — Hydrogen  peroxide   is  a  valuable 
germicide,  applicable  to  the  cleansing  of  mucous  surfaces  and  wounds. 
It  is  readily  reduced  to  H2O  and  nascent  oxygen  in  contact  with 
organic  substances,   and  its  efficiency  is  attributable  to  the  latter 
element.    It  is  essential  that  the  peroxide  actually  reach  the  organism 
to  be  destroyed  in  order  to  be  effective.    Usually  hydrogen  peroxide 
is  quite  acid  in  reaction  and  irritating  for  this  reason. 

3.  Chlorinated  Lime  or  "  Bleach." — Chlorinated  lime  is  an  excellent 
deodorant  and  germicide  when  it  is  fresh,  but  it  soon  loses  chlorine 
when  exposed  to  the  air.    Nascelit  chlorine  is  liberated  from  aqueous 
solutions,  and  reacts  with  water  to  form  nascent  oxygen  and  hydro- 
chloric acid,  according  to  the  equation  2C1  +  H^O  =  2HC1  +  O. 


246          STERILIZATION,  ANTISEPSIS  AND  DISINFECTION 

One  part  of  nascent  chlorine  to  1,000,000  parts  of  water— a  milligram 
to  a  litre  in  other  words — will  kill  moderate  numbers  of  bacteria 
within  a  few  minutes.  For  this  reason,  chlorinated  lime  is  extensively 
used  in  the  treatment  of  swimming  pools  to  reduce  the  bacterial  count. 
It  is  also  used  for  the  practical  sterilization  of  urine,  bath  water,  feces, 
and  in  the  solid  state,  in  privies,  cellars,  and  stables. 

Phenols,  Cresols. — Phenol,  popularly  known  as  carbolic  acid,  and 
cresols,  of  which  three  are  known — ortho,  meta,  and  para — are  powerful 
germicides : 

OH  OH  OH  OH 

0        0 

\/CHs  \/ 

CH3 
Phenol  Ortho  cresol  Meta  cresol  Para  cresol 

Phenol  and  the  cresols  are  somewhat  sparingly  soluble  in  water. 
A  6  per  cent,  aqueous  solution  of  carbolic  acid,  and  5  per  cent,  solu- 
tions of  the  cresols  are  about  the  limits  of  solubility;  3  to  5  per  cent, 
solutions  are  used  for  most  practical  purposes.  Phenol  and  cresols 
are  not  only  very  toxic  for  bacteria,  they  are  caustic  and  poisonous 
for  human  tissue  as  well.  Stronger  solutions  are  anesthetic,  sugges- 
tive of  a  definite  action  upon  nervous  tissue.  These  substances  appear 
to  be  readily  absorbed  from  mucous  surfaces,  the  skin,  and  wounds. 
They  are  excreted,  in  part  at  least,  through  the  kidneys.  "Smoky 
urine,"  indicating  an  irritation  of  the  kidney  tissue,  is  a  not  uncommon 
sequel  of  carbolic  acid  poisoning. 

A  3  per  cent,  solution  of  phenol  is  approximately  equivalent  in  its 
disinfectant  value  to  a  1  to  1000  solution  of  bichloride  of  mercury, 
but  it  does  not  unite  readily  with  proteins  to  form  insoluble,  inert 
compounds,  and  it  is  not  destructive  of  fabrics,  metals  and  articles 
of  every-day  use.1  For  sputum,  urine,  feces,  purulent  discharges,  and 
for  stained  and  soiled  linen,  a  5  per  cent,  solution,  equal  in  volume  to 
the  bulk  of  the  material  to  be  disinfected,  is  used  and  allowed  to  remain 
at  least  one  hour  before  being  disturbed. 

Cresols  form  soaps  with  caustic  solutions,  which  are  strongly  ger- 
micidal.  An  excellent  cresol  soap  may  be  made  by  adding  one  part 
by  volume  of  cresols  to  an  equal  amount  of  soft  soap  (potash  soap). 
This  is  stirred  thoroughly  and  allowed  to  stand  twenty-four  hours. 
A  5  per  cent,  aqueous  solution  of  this  preparation  is  nearly  three  times 
as  efficient  in  its  disinfectant  value  as  a  5  per  cent,  solution  of  carbolic 
acid. 

1  Hamilton,  Therapeutic  Gazette,  1914,  xxxviii,  311. 


LABORATORY  STERILIZATION  247 

Tincture  of  lodin. — In  vitro,  tincture  of  iodin  is  of  little  value  as  a 
germicide,  but  freshly  prepared  tincture  of  iodin  applied  to  the  skin 
appears  to  possess  very  considerable  germicidal  value.  This  solution 
seems  to  be  most  effective  when  it  is  freshly  prepared  and  works  most 
satisfactorily  when  the  part  upon  which  it  is  to  be  used  has  been 
cleaned  with  alcohol  and  allowed  to  dry.  Nascent  iodin  is  liberated, 
and  it  is  stated  that  iodin  in  statu  nascendi  is  the  active  germicidal 
factor.  Tincture  of  iodin  is  rather  widely  used  as  a  skin  disinfectant 
for  minor  operations,  for  sterilizing  the  epidermis  prior  to  spinal 
puncture,  collecting  blood  for  cultural  purposes,  and  for  operations 
upon  laboratory  animals.  Iodin  is  absorbed  through  the  skin,  and 
in  large  amounts  it  is  toxic. 

Boric  Acid.— Boric  acid  is  frequently  used  upon  mucous  surfaces 
and  other  exposed  parts  when  a  very  mild  antiseptic  solution  is 
required.  Boric  acid  is  rather  an  antiseptic  than  a  germicide:  its 
chief  advantage  lies  in  the  fact  that  1  to  3  per  cent,  aqueous  solutions 
have  but  little  action  on  the  tissues. 

All  disinfectants  appear  to  be  cellular  poisons  to  a  greater  or  lesser 
degree;  in  lesser  concentrations  they  are  without  marked  effect  upon 
microorganisms;  in  effective  concentrations  they  appear  to  form 
combinations  with  tissues  if  they  are  used  in  or  on  man. 

Disinfection  of  the  tissues  has  been  attempted  with  specific  bac- 
tericidal sera,  which  are  without  noteworthy  harmful  effects  upon 
the  patient.  At  the  present  time  immune  sera  are  not  wholly  satis- 
factory for  this  purpose,  but  sufficiently  encouraging  results  have 
been  obtained  to  justify  their  present  use  and  to  afford  promise  of 
their  improvement  in  the  future. 

A  majority  of  chemical  disinfectants  are,  to  use  Ehrlich's  termin- 
ology* organotrophic  rather  than  parasitotrophic,  that  is,  they  have  a 
greater  affinity  for  the  tissues  of  the  host  than  for  the  parasite.  Quinine, 
on  the  contrary,  appears  to  be  parasitotrophic — it  is  almost  a  specific 
for  malarial  parasites.  Ehrlich's  brilliant  researches  in  chemotherapy 
have  added  organic  compounds  containing  arsenic  to  the  list  of  para- 
sitotrophic substances;  they  have  a  very  direct  and  inimical  action 
upon  trypanosomes  and  the  Treponemata,  and  but  minimal  action 
upon  the  tissues  of  the  host. 

Formaldehyde. — A  solution  of  formaldehyde  gas  in  water,  commer- 
cially known  as  formalin,  is  a  powerful  disinfectant;  it  does  not  react 
as  strongly  as  mercuric  chloride  with  protein  solutions;1  it  does  not 

1  Formaldehyde  unites  with  ammonia  and  with  the  amino-nitrogen  of  amino  acids  to 
form  stable  compounds;  there  is  relatively  little  action  upon  native  proteins,  however. 


248          STERILIZATION,  ANTISEPSIS  AND  DISINFECTION 

injure  metals  or  ordinary  fabrics.  The  commercial  solution  con- 
tains about  35  per  cent,  of  formaldehyde,  hence  a  10  per  cent,  solu- 
tion of  "formalin"  will  contain  but  3.5  per  cent,  of  " formaldehyde," 
which  is,  of  course,  the  reactive  substance.  Formaldehyde  is  an 
excellent  disinfectant  for  sputum,  urine  and  feces,  and  other  excre- 
tions; a  5  per  cent,  solution  of  formalin  (corresponding  to  about  2 
per  cent,  formaldehyde)  in  the  proportion  of  two  volumes  of  the  disin- 
fectant to  one  of  the  excretion  will  effect  practical  sterilization  of 
feces  within  an  hour.  Fomites  are  sterilized  in  the  same  manner. 
The  fumes  are  irritating,  and  disinfection  should  not  be  practiced 
in  the  sick-room. 

Essential  Oils. — Essential  oils  have  been  used  extensively  in  the 
past,  particularly  in  the  treatment  of  nasal  and  pharyngeal  infections, 
and  for  mouth-washes.  Menthol,  thymol  and  eucalyptol,  the  active 
principles  of  oil  of  peppermint,  thyme  and  eucalyptus  respectively, 
undoubtedly  possess  antiseptic  and  feebly  germicidal  properties. 
Cloves,  cinnamon  and  other  spices  have  been  used  for  the  preserva- 
tion of  certain  types  of  foods;  their  efficiency  probably  depends  largely 
upon  their  content  of  essential  oils.  The  expense  of  these  substances 
compared  with  their  efficiency  as  antiseptics  makes  their  use  practically 
prohibitive. 

Soaps. — Cleanliness  is  a  very  important  barrier  to  the  spread  of  dis- 
ease. Very  few  pathogenic  bacteria  upon  exposed  surfaces  of  rooms 
can  survive  an  application  of  hot  soap  suds  applied  with  a  vigorous 
arm  and  a  scrubbing  brush.  A  5  per  cent,  solution  of  washing  soda 
(commercial  sodium  carbonate)  is  even  more  efficient  if  applied  hot, 
but  there  are  limitations  to  its  use.  Fine  furnishings  and  hangings, 
wall  paper  and  similar  objects  cannot  ordinarily  be  treated  with  liquid 
disinfectants. 

Testing  and  Standardizing  Liquid  Disinfectants. — The  first  satis- 
factory method  of  comparing  the  disinfectant  value  of  chemical  disin- 
fectants was  that  of  Rideal  and  Walker,1  widely  known  as  the  "Car- 
bolic Coefficient"  method.  A  modification  of  this  method,  proposed 
by  Anderson  and  McClintic,2  is  widely  used  in  the  United  States. 
Briefly,  the  method  as  modified  by  Anderson  and  McClintic  consists 
in  comparing  the  activity  of  the  unknown  disinfectant  solution  in 
various  dilutions  with  a  standard  solution  of  carbolic  acid;  Bacillus 


1  Jour.  Sanitary  Institute,  London,  xxiv. 

2  Bull.  Hyg.  Lab.,  Washington,  D.  C.,  April,  1912,  No.  82.    Full  details  of  method  and 
the  disinfectant  value  of  a  large  number  of  substances  are  given. 


LABORATORY  STERILIZATION  249 

typhosus  is  the  organism  selected  for  the  purpose,  and  the  strength 
of  solution  of  both  the  unknown  and  known  solutions  are  carefully 
measured.  The  time  and  temperature  of  exposure  of  the  organism  to 
the  disinfectant  solutions  and  the  nature  of  the  medium  in  which  the 
exposure  is  made  are  carefully  controlled.  Even  with  the  most  rigor- 
ous attention  to  details,  the  carbolic  coefficient  of  the  same  disinfectant 
determined  by  this  method  varies  nearly  50  per  cent,  in  the  hands  of 
different  observers;1  for  the  present,  the  standards  of  the  Public 
Health  and  Marine  Hospital  Service2  are  regarded  as  official  for  the 
United  States. 

Gaseous  Disinfectants. — Pathogenic  bacteria  which  are  known  or 
suspected  to  be  present  upon  fabrics  or  furnishings  injured  by  chemical 
disinfectant  solutions,  as  well  as  bacteria  promiscuously  distributed 
in  rooms  through  droplet  infection  and  by  dust  may  be  killed  by 
gaseous  disinfectants,  of  which  several  are  available. 

1 .  Formaldehyde. — Formaldehyde  is  the  most  efficient  of  the  gaseous 
disinfectants  for  superficial  disinfection,  but  its  limited  power  of 
penetration  must  be  borne  in  mind.  Formaldehyde  is  dispensed  com- 
mercially under  the  name  "formalin,"  which  signifies  a  40  per  cent, 
volume  solution  of  the  gas  (formaldehyde)  in  water.  Commercial 
formalin  rarely  contains  more  than  36  per  cent,  of  formaldehyde  by 
volume,  however,  and  in  practice  it  is  well  to  estimate  35  per  cent,  as 
a  working  basis.  Commercial  solutions,  it  must  be  remembered,  are 
always  acid,  and  the  gas  itself  in  small  amounts  is  irritating  to  mucous 
membranes.  Prolonged  exposure  to  concentrations  of  the  gas  suffi- 
cient to  kill  bacteria  may  be  fatal  to  animals.  The  gas  has  practically 
no  insecticidal  value.  In  sufficient  concentration  the  gas  is  inflam- 
mable and  may  be  ignitecrby  any  free  flame. 

In  the  past  formaldehyde  was  liberated  from  its  aqueous  solution 
in  the  gaseous  state  in  complicated  retorts,  autoclaves  or  lamps  of 
special  design.  Much  simpler  methods  have  been  evolved,  which  are 
now  used  almost  exclusively  in  practical  gaseous  disinfection.  Of 
these  the  permanganate  method  and  the  "  sheet- volatilization  method" 
are  the  most  widely  used;  the  former  possesses  the  dual  advantage 
of  a  quick  liberation  of  the  entire  available  amount  of  disinfectant, 
and  very  simple  apparatus ;  the  latter  is  advantageous  when  a  gradual 
evolution  of  gas  and  a  prolonged  exposure  to  its  action  are  desired. 

The  Permanganate  Method. — When  formalin  is  poured  upon  crystals' 
of  potassium  permanganate,  an  energetic  reaction  with  the  evolution 

1  Hamilton  and  Ohno,  Jour.  Pub.  Health,  1913;  ibid.,  1914,  iv,  163. 

2  Bull.  Hyg.  Lab.,  Washington,  D.  C.,  April,  1912,  No.  82. 


250          STERILIZATION,  ANTISEPSIS  AND  DISINFECTION 

of  sufficient  heat  to  boil  the  liquid  takes  place.  Formaldehyde  gas  and 
heated  water  vapor  are  evolved.  The  entire  process  requires  but  a 
few  minutes,  and  when  two  parts  of  formalin  to  one  part  of  perman- 
ganate are  used  the  residue  is  small  in  amount  and  practically  dry 
and  free  from  reactive  substances. 

Ten  ounces  of  formalin  and  five  ounces  of  permanganate  of  potash 
crystals  are  required  for  each  thousand  cubic  feet  of  space  to  be  disin- 
fected. The  temperature  must  be  not  less  than  60°  F.,  and  the 
humidity  must  be  at  least  60  per  cent,  for  successful  results.  It  is 
convenient  to  place  the  permanganate  in  a  three-gallon,  galvanized- 
iron  pail  with  flaring  sides,  because  the  reaction  between  permanganate 
and  formalin  is  attended  with  considerable  spattering.  It  is  also 
advisable  to  place  two  or  three  layers  of  heavy  paper  under  the  pail, 
of  sufficient  size  to  project  two  feet  at  least  in  all  directions,  or  better, 
to  place  a  galvanized-iron  plate  of  similar  dimensions  under  the  pail 
to  catch  all  the  liquid  which  is  ejected  from  the  pail  during  the  process 
of  evolution  of  the  gas.  For  successful  disinfection,  all  closets,  drawers 
and  alcoves  should  be  opened  as  freely  as  possible;  doors,  windows 
and  fireplaces  leading  to  the  exterior  should  be  tightly  closed.  The 
room  should  be  left  closed  and  undisturbed  for  at  least  four  hours. 

The  Sheet  Volatilization  Method. — This  method  requires  no  appara- 
tus except  sheets,  and  some  mechanical  device  for  spraying  formalin 
upon  the  sheets.  The  conditions  of  moisture  and  humidity  and  the 
same  general  preparation  of  the  room  as  for  the  potassium  perman- 
ganate formalin  method  must  prevail. 

Sheets  are  hung  upon  tightly  stretched  cords  or  other  similar  sup- 
port, in  such  a  manner  that  they  rest  at  an  angle  of  about  45°  with 
the  perpendicular.  They  are  wet  with  warm  water,  are  "wrung  out" 
to  remove  the  excess,  and  sprayed  with  formalin  in  the  proportion  of 
ten  ounces  to  each  thirty  square  feet  of  surface.  One  sheet  (thirty 
feet  square)  is  sufficient  for  each  thousand  cubic  feet  of  room  space. 

The  evolution  of  formaldehyde  is  slower  with  the  sheet  method  than 
with  the  permanganate  method,  but  equally  efficient  disinfection  is 
obtained  if  the  room  is  kept  closed  eight  hours. 

2.  Paraform. — Paraform  is  a  polymer  of  formaldehyde;  it  is  a  white 
solid  which  is  readily  ignited,  and  burns  with  a  bluish  flame.  It  offers 
no  advantages  over  formaldehyde,  except  that  it  occupies  much  less 
space.  Special  lamps  have  been  devised  to  liberate  formaldehyde 
from  it  in  the  gaseous  state,  but  the  efficiency  of  the  method  is  not 
greater  than  the  permanganate  method,  and  the  apparatus  is  some- 


LABORATORY  STERILIZATION  251 

what  more  expensive,  and  bulky  to  transport.  Paraform  dissolved 
in  warm  wrater,  in  the  proportion  of  two  ounces  of  the  former  to  half 
a  pint  of  the  latter  may  be  used  in  place  of  formalin  either  in  the 
permanganate  method  or  the  volatilization  method  described  in  the 
foregoing. 

Attempts  have  been  made  to  combine  paraform  and  sulphur  in  the 
form  of  candles  or  pastilles  for  purposes  of  disinfection.  Such  pre- 
parations are  valueless  so  far  as  the  generation  of  formaldehyde  is 
concerned,  because  the  products  of  combustion  of  this  substance  are 
carbon  dioxide  and  water. 

3.  Sulphur. — Sulphur  was  formerly  highly  regarded  as  a  gaseous 
disinfectant,  but  it  is  now  used  chiefly  for  insecticidal  fumigation". 
The  products  of  combustion  are  SO2  and  SO3,  both  gases;  in  the 
presence  of  moisture  they  have  considerable  germicidal  activity,  but 
little  penetrating  power. 

Sulphur  dioxide  and  trioxide  are  vigorous  bleaching  agents;  they 
destroy  fabrics,  fine  furnishings,  and  are  injurious  to  painted  or  var- 
nished surfaces.  Consequently,  the  usefulness  of  sulphur  as  a  germi- 
cide is  restricted  to  the  holds  of  ships,  to  warehouses  and  similar  struc- 
tures, where  the  destruction  of  vermin  is  an  important  factor  in  the 
disinfecting  process. 

At  least  5  pounds  of  sulphur  for  each  1000  cubic  feet  of  space  to  be 
disinfected  are  placed  in  a  broad,  shallow  iron  pot,  preferably  from 
one  to  two  feet  in  diameter  and  from  three  to  six  inches  high.  These 
are  placed  in  pans  containing  about  two  inches  of  water,  both  to 
prevent  damage  if  the  pot  cracks  during  the  burning  process,  and  to 
supply  moisture  essential  to  the  success  of  the  disinfection.  The 
sulphur  should  be  not  more  than  three  inches  deep  in  the  pot  and 
should  slope  gently  from  the  edges  of  the  pot  to  the  center,  where 
a  crater  is  hollowed  out  and  filled  with  an  ounce  of  alcohol  to  start 
combustion.  The  sulphur  burns  slowly,  and  all  cracks,  doors  and 
windows  should  be  sealed  with  paper  and  paste  to  prevent  escape  of 
the  fumes.  At  least  twelve  hours  should  be  allowed  before  the  room 
is  opened. 

Liquid  sulphur  dioxide  is  sometimes  used  in  place  of  burning  sul- 
phur; the  cost  is  several  times  that  of  burning  sulphur,  and  for  the 
practical  disinfection  of  rooms  it  is  rarely  used. 

4.  Chlorine  Gas. — Chlorine  gas,  particularly  in  humid  atmospheres, 
possesses  considerable  germicidal  power,  but  its  extremely  corrosive 
action  upon  fabrics  and  furnishings  has  materially  restricted  its  field 
of  usefulness  for  practical  disinfection. 


252          STERILIZATION,  ANTISEPSIS  AND  DISINFECTION 

5.  Ozone. — Nascent  oxygen  in  actual  contact  with  bacteria  is  a 
powerful  germicide,  and  aside  from  the  cost  of  production,  it  is  of 
value  for  the  purification  of  water  for  domestic  purposes.  As  an  aerial 
disinfectant,  however,  it  has  been  disappointing. 

PRACTICAL   DISINFECTION. 

Sputum. — The  bacteria  and  other  microorganisms  which  incite 
disease  of  the  mouth,  nose  and  respiratory  tract  leave  the  patient 
chiefly  in  the  nasal  secretion  and  sputum.  They  are  eliminated  in 
"droplets"  of  sputum  during  violent  expulsion  of  the  expired  air,  as 
in  coughing  and  sneezing.  The  patient,  therefore,  should  be  instructed 
to  cough  or  sneeze  into  paper  or  cloth  napkins,  to  prevent  the  escape 
of  infected  droplets  of  sputum,  and  to  expectorate  into  a  sputum  box 
provided  with  a  cover.  The  paper  napkins  should  be  placed  in  a 
covered  receptacle  and  eventually  burned.  Cloth  napkins  may  be 
satisfactorily  treated  by  complete  immersion  in  boiling  water  for  at 
least  fifteen  minutes. 

Sputum  may  be  disinfected  with  5  per  cent,  carbolic  or  cresol  solu- 
tion, or  with  a  5  per  cent,  solution  of  formalin.  At  least  one  hour's 
exposure  to  the  disinfectant  is  required. 

Vomitus. — An  elimination  of  pathogenic  bacteria  from  the  body 
in  vomitus  is  by  no  means  impossible,  although  relatively  little  atten- 
tion has  been  paid  to  this  subject  in  the  past.  The  cholera  vibrio  s 
probably  the  most  formidable  organism  to  be  reckoned  with,  but  the 
possibility  of  typhoid  bacilli  must  be  borne  in  mind.  Vomitus  should 
be  handled  with  the  same  precautions  as  infected  feces. 

Feces  and  Urine. — Those  organisms  which  are  the  etiological  agents 
of  infections  involving  the  gastro-intestinal  tract,  as  typhoid,  dysen- 
tery, paratyphoid  bacilli  and  cholera  vibrios,  amoebae,  and  probably 
the  unknown  excitants  of  the  intestinal  disorders  escape  from  the 
diseased  host  chiefly  in  the  feces,  and  occasionally  in  the  urine. 

The  feces  and  urine  should  be  received  in  porcelain  or  metal  con- 
tainers of  appropriate  pattern  to  prevent  mechanical  loss  of  material 
and  immediately  mixed  with  twice  the  volume  of  carbolic  acid  or 
cresol  solution,  an  equal  volume  of  5  per  cent,  formalin  solution,  or 
with  chloride  of  lime  in  the  proportion  of  10  per  cent,  of  the  total 
volume  of  feces  and  urine.  The  fecal  mass,  unless  completely  fluid, 
should  be  intimately  mixed  with  the  disinfectant  solution  and  allowed 
to  remain  in  contact  with  it  at  least  an  hour.  The  soiled  parts  of  the 


PRACTICAL  DISINFECTION  253 

patient  should  be  wiped  with  a  cloth  dipped  in  2  per  cent,  carbolic 
acid  or  cresol  solution,  then  with  water  to  remove  the  disinfectant. 
The  cloths  should  be  either  placed  at  once  in  briskly  boiling  water,  or 
in  the  bedpan,  and  treated  with  the  feces. 

Fomites. — Soiled  linen,  clothing  and  bedding  should  be  immersed 
in  a  liberal  amount  of  2  or  3  per  cent,  carbolic  acid  solution  and  left 
at  least  two  hours.  An  exposure  of  fifteen  minutes  in  briskly  boiling 
water,  provided  a  considerable  volume  is  used,  is  also  sufficient  to 
disinfect  soiled  fomites. 

Bath  Water. — The  water  in  which  patients  suffering  from  intestinal 
infections  have  bathed  should  be  disinfected  before  it  is  discharged 
into  a  drain.  An  ounce  of  chlorinated  lime  thoroughly  mixed  with 
the  bath  water  will  disinfect  it  within  an  hour.  The  sides  of  the  bath- 
tub above  the  level  of  the  water  must  be  disinfected  as  well  as  the 
water  itself. 

Skin  and  Hands. — Infection  of  the  skin  and  -hands,  both  of  the 
patient  and  attendants,  is  frequently  unavoidable  in  intestinal  diseases. 
A  vigorous  application  of  a  scrubbing  brush  and  green  soap  and  a 
thorough  cleansing  of  the  nails  frequently  suffices  for  the  hands.  An 
application  of  2  to  3  per  cent,  carbolic  acid,  or  1  to  1000  bichloride  of 
mercury  for  several  minutes  will  remove  all  danger  of  infection. 

Sterilization  of  the  hands  for  surgical  operations  is  still  a  subject 
of  debate;  there  is  little  uniformity  in  the  methods  advocated  by 
leading  surgeons.  Wearing  sterilized  rubber  gloves  during  operations 
is  a  common  practice. 

Instruments. — The  preparation  of  instruments  for  surgical  use, 
often  erroneously  called  "sterilization,"  must  be  sharply  distinguished 
from  true  sterilization  in  the  bacteriological  sense.  Simple  boiling  of 
surgical  appliances  in  soda  solution  does  not  necessarily  render  them 
free  from  bacterial  spores,  although  the  method  is  efficient  for  surgical 
technic  because  the  residual  bacteria  which  may  survive  this  treatment 
do  not  germinate  in  the  tissues.  It  is  frequently  deemed  sufficient  to 
boil  syringes  and  other  appliances  used  for  removing  blood  or  other 
material  for  bacteriological  study;  the  only  trustworthy  method  for 
this  purpose  is  the  autoclave  or  the  hot-air  sterilizer,  depending  upon 
the  nature  of  the  appliance. 

The  use  of  carbolic  acid  is  not  recommended  for  bacteriological 
syringes  and  other  apparatus  used  in  collecting  material  for  bacterio- 
logical examination;  it  is  difficult  to  remove  the  last  traces  of  the  disin- 
fectant without  contaminating  the  instrument  itself. 


254          STERILIZATION,  ANTISEPSIS  AND  DISINFECTION 

Clinical  Thermometers,  Dental  Instruments. — Clinical  ther- 
mometers and  dental  instruments  are  ethically  on  a  par  with  the 
common  drinking  cup  and  the  common  towel.  Barbers'  razors  and 
brushes  also  belong  to  this  group.  The  cost  of  such  instruments  is 
prohibitive  for  individual  use,  however,  and  their  disinfection  appears 
to  be  the  practical  solution  of  the  problem.  In  hospitals  the  ther- 
mometers can  be  sterilized  readily,  first,  by  wiping  them  carefully 
to  remove  adherent  mucus,  then  immersing  them  in  5  per  cent,  for- 
malin solution,1  where  they  remain  until  wanted  again.  A  thorough 
rinsing  in  water  will  remove  the  formalin.  The  clinician  who  has  an 
extensive  visiting  practice  cannot  afford  individual  thermometers; 
for  practical  purposes  his  thermometer  can  be  kept  free  from  bacteria 
if  it  is  washed  each  time  in  running  water  until  free  from  mucus,  and 
kept  in  a  metallic  case  containing  10  per  cent,  formalin  solution  pre- 
pared daily.  Running  water  will  remove  all  traces  of  formalin  before 
use.  At  least  two  hours  should  be  allowed  before  sterilization  is 
regarded  as  complete.  Several  thermometers  may  be  required  to 
permit  of  this  period  of  sterilization  for  each  individual  instrument. 

Dentists'  instruments  almost  without  exception  can  be  safely 
sterilized  in  a  boiling  5  per  cent,  solution  of  washing  soda  (sodium 
carbonate)  within  five  minutes'  exposure.  If  they  are  then  wiped 
dry  there  is  little  danger  of  rusting.  The  sterilization  of  dental  mouth 
mirrors  is  a  problem  which  would  appear  to  require  special  investiga- 
tion. 

1  A  covered  container  is  required;  the  fumes  of  formaldehyde  are  very  irritating  to 
the  patient. 


SECTION    II. 

PATHOGENIC  BACTERIA. 


CHAPTER  XII. 
THE  PYOGENIC  COCCI. 


THE  BACTERIA  OF  INFLAMMATION. 
THE  STAPHYLOCOCCUS  GROUP. 
Micrococcus  Aureus. 


Staphylococcus   Pyogenes  Albus. 
Staphylococcus  Epidermidis  Albus. 
Micrococcus   Tetragenus. 


Staphylococcus  Pyogenes  Citreus.        ;      Micrococcus  Ovalis. 

THE   BACTERIA   OF  INFLAMMATION. 

THERE  is  a  group  of  bacteria  which  possesses  in  common  the  ability 
to  incite  that  type  of  infection  which  is  commonly  spoken  of  as  inflam- 
mation. A  majority  of  these  organisms  are  habitual  parasites  of  man 
living  upon  the  exposed  surfaces  of  the  body,  the  skin  and  mucous 
membranes  chiefly:  with  respect  to  their  pathogenic  properties  they 
may  be  regarded  as  "opportunists,"  not  as  a  rule  requiring  a  well- 
defined  portal  of  entry  through  definite  tissues  to  become  invasive. 
Any  break  in  the  continuity  of  the  skin  or  a  weakening  or  change  in 
the  physiological  state  of  a  mucous  membrane  (frequently  caused  by 
intracurrent  infection)  provides  the  necessary  atrium  for  invasion  of 
the  underlying  tissues. 

Not  only  are  these  bacteria  ordinarily  unable  of  themselves  to 
locate  and  force  an  entrance  to  the  tissues  of  their  host;  after  invasion 
is  accomplished  they  are  unable  to  escape  from  the  tissues  in  suffi- 
cient numbers  to  cause  progressive  disease  of  like  nature  in  other 
hosts.  They  are  locked  up  in  the  body,  as  it  were,  and  eventually 
perish.  They  have  not  perfected  their  pathogenic  mechanism.  (See 
chapter  on  Parasitism.) 

Bacteria  of  the  "opportunist"  type  may  be  raised  to  very  con- 
siderable pathogenic  powers  if  artificially  created  atria  of  entrance  to 
and  escape  from  the  tissues  are  provided,  as  for  example,  by  passage 


256  THE  PYOGENIC  COCCI 

through  suitable  animals,  but  they  soon  tend  to  lose  their  artificially 
acquired  pathogenic  properties  under  ordinary  conditions  and  return 
again  to  a  parasitic  existence. 

Prominent  among  these  habitually  parasitic  bacteria  which  occur 
on  the  skin  and  mucous  membranes  of  man  are  the  various  members 
of  the  Staphylococcus  and  Streptococcus  Groups. 

THE   STAPHYLOCOCCUS   GROUP. 

Micrococcus  Aureus. — Synonyms. — Staphylococcus  pyogenes  aureus; 
Staphylococcus  aureus;  Micrococcus  pyogenes  aureus;  Micrococcus  sali- 
varius  aureus. 

Historical. — Staphylococci  probably  were  first  seen  by  Klebs,  some- 
what later  by  Billroth,  in  unstained  pus.  Pasteur1  repeatedly  isolated 
them  from  the  pus  of  furuncles,  and  in  one  case  of  osteomyelitis,  and 
suggested  their  etiological  relationship  to  these  lesions,  but  to  Rosen- 
bach2  belongs  the  priority  of  growing  them  in  cultures  of  undoubted 
purity. 

Morphology. — The  organisms  in  the  free  state  are  spherical,  measur- 
ing from  0.7  to  0.9  micron  in  diameter.  Those  just  about  to  divide 
are  frequently  oval.  They  occur  singly,  in  pairs,  or  in  irregular  masses, 
both  in  culture  and  in  pus;  rarely  chains  of  four  to  six  cocci  are  found. 

Staphylococci  are  non-motile  and  possess  no  flagella;  they  do  not 
form  capsules,  and  spores  have  not  been  observed.  They  siain  readily 
with  ordinary  anilin  dyes,  some  individuals  more  intensely  than  their 
fellows.  They  are  Gram-positive. 

Isolation  and  Culture. — Staphylococci  are  readily  obtained  in  pure 
culture  by  plating  or  streaking  the  suspected  material  directly  upon 
agar  or  gelatin.  The  colonies  on  gelatin  after  thirty  to  forty-eight 
hours'  incubation  at  room  temperature  become  visible  as  gray,  glis- 
tening growths  0.5  to  1  mm.  in  diameter;  somewhat  later  the  colonies 
sink  into  saucer-shaped  depressions  of  liquefied  gelatin,  the  bacteria 
collect  at  the  bottom  of  the  depression  and  soon  become  golden-yellow 
in  color.  The  growth  upon  agar  plates  at  37°  C.  is  more  rapid:  at 
the  end  of  forty-eight  hours'  incubation  the  colonies  are  golden-yellow 
and  have  attained  a  diameter  of  1  to  3  mm. 

Staphylococci  grow  readily  in  the  ordinary  cultural  media.  Gela- 
tin, coagulated  blood  serum  (sugar-free)  and  casein  are  liquefied. 

1  Compt.  rend.  Acad.  Sci.,  1880,  xc,  1035. 

2  Mikroorganismen  bei  den  Wundinfektionskrankheiten  des  Menschen,   Wiesbaden, 
1884,  19. 


THE  STAPHYLOCOCCUS  GROUP  257 

Acid  is  produced  in  dextrose,  lactose,  saccharose  and  mannite  broths. 
Milk  is  coagulated,  usually  within  three  days  at  37°  C.;  many  strains 
subsequently  partially  digest  the  coagulum.  In  plain  and  dextrose 
broths  a  turbidity  is  produced  after  twelve  to  fourteen  hours'  incuba- 
tion at  37°  C.;  after  forty-eight  hours'  growth  a  golden-yellow  sedi- 
ment collects  in  the  bottom  of  the  tubes. t  Growth  on  slanted  agar  is 
golden-yellow  in  color,  moist  and  spreading.  Pigment  production  is 
especially  luxuriant  on  slanted  potato. 

The  organisms  are  aerobic,  facultatively  anaerobic.  The  optimum 
temperature  of  growth  lies  between  28°  and  38°  C.;  growth  ceases 
below  8°  C.  and  above  43°  C. 


FIG.  32.— Staphylococcus.      X  1000. 

Staphylococci  are  among  the  most  resistant  of  the  non-spore-form- 
ing bacteria  to  physical  agents.  An  exposure  of  one  hour  at  80°  C 
or  two  hours  at  70°  C.  moist  heat  is  usually  fatal.  Several  minutes' 
exposure  at  100°  C.  (flowing  steam)  or  twelve  hours'  exposure  to  direct 
sunlight  may  fail  to  kill  them.  Indirect  daylight  may  fail  to  destroy 
their  vitality  even  after  two  weeks;  three  months'  continuous  drying 
(on  cloth  or  paper)  is  equally  ineffective;  0.001  per  cent,  mercuric 
chloride  and  5  per  cent,  carbolic  acid  usually  kill  the  naked  germs  in 
about  ten  minutes. 

Products  of  Growth. — Acids,  chiefly  lactic,  but  with  demonstrable 
amounts  of  propionic,  butyric,  and  valerianic,  are  formed  during  the 
fermentation  of  ordinary  sugars.  No  gas  is  produced.  The  pus  of 
Staphylococcus  abscesses  is  usually  acid  in  reaction;  the  organisms 
appear  to  form  limited  amounts  of  acid  from  protein.1  Emmering2 

1  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Assn.,  1913,  xxxv,  1246. 

2  Berlin,  deut.  chem.  Gesellsch.,  1896,  2721. 
17 


258  THE  PYOGENIC  COCCI 

has  identified  indol,  phenol,  skatol,  and  trimethylamine  among  the 
decomposition  products  of  staphylococci  grown  anaerobically  in 
protein  media.  Cacace1  has  shown  that  the  earlier  decomposition 
products  produced  from  gelatin  and  coagulated  blood  serum  are 
chiefly  proteoses  and  peptones;  as  proteolysis  proceeds,  these  products 
are  degraded  to  simpler  amino  acid  compounds. 

Pigment. — Staphylococci  isolated  directly  from  severe  inflamma- 
tions usually  produce  a  golden-yellow  pigment,  but  prolonged  cul- 
tivation upon  artificial  media  may  result  in  a  partial  or  complete  loss 
of  chromogenesis.  Armand2  has  isolated  non-chromogenic  strains 
of  staphylococci  directly  from  typically  chromogenic  cultures  by  the 
plate  method.  The  yellow  pigment,  which  is  produced  most  abun- 
dantly in  media  containing  carbohydrates  (particularly  on  potato) 
in  the  presence  of  free  oxygen,  appears  to  lie  between  the  individual 
organisms,  not  within  their  substance.  It  is  insoluble,  or  nearly  so, 
in  water,  readily  soluble  in  alcohol.  It  is  related  to  the  lipochromes. 
The  pigment  can  be  saponified  readily,  and  it  evolves  an  odor  of  acro- 
lein  when  it  is  dry-heated.  Strong  acids,  notably  sulphuric,  change 
the  yellow  color  to  a  green-blue  (lipocyanin) .  Lugol's  solution 
(iodin-potassium  iodide)  turns  it  green. 

Enzymes. — 1.  Proteolytic. — Old  sugar-free  broth  and  gelatin  cul- 
tures of  staphylococcus  contain  a  proteolytic  enzyme  which  will  liquefy 
gelatin — a  gelatinase.  This  enzyme  may  be  obtained  in  an  active 
state  free  from  bacteria  by  filtering  either  broth  or  liquefied  gelatin 
cultures  of  the  organism  through  unglazed  porcelain.3  An  enzyme 
which  liquefies  casein  is  demonstrable  in  milk  cultures;  whether  the 
latter  enzyme  is  identical  with  the  gelatinase  has  not  been  determined. 

2.  Amylolytic. — According   to    Buxton,4    staphylococci    produce    a 
maltase  which  hydrolyzes  maltose;  no  other  inverting  enzymes  have 
been  observed. 

3.  Lipolytic. — Wells  and  Corper5  have  demonstrated  a  lipase  of 
moderate  activity  in  autolyzed  agar  slant  cultures  of  staphylococci. 

4.  Hemolytic. — Neisser6    and    Wechsberg7    have    shown    that    old 
(7-  to  14-day)  broth  cultures  of  staphylococci,  particularly  the  more 
virulent  strains,  contain  a  soluble  enzyme  which  hemolyzes  blood 

1  Cent.  f.  Bakt.,  1901,  xxx,  244. 

2  Quoted  by  Lehmann  and  Neumann,  Bacteriology,  1904,  3d  ed.,  193. 

3  Loeb,  Cent.  f.  Bakt.,  1902,  xxxii,  471. 

4  Am.  Med.,  1903,  vi,  137. 

s  Jour.  Inf.  Dis.,  1912,  xi,  388. 

6  Zeit,  f.  Hyg.,  1901,  xxxvi,  299. 

7  Cent.  f.  Bakt.,  Orig.,  1903,  xxxiv,  857. 


THE  STAPHYLOCOCCUS  GROUP  259 

both  in  vivo  and  in  vitro.  In  vitro  this  enzyme,  staphylolysin,  appears 
to  digest  the  stroma  of  red  blood  cells,  liberating  hemoglobin  from 
them.  A  quantitative  measure  of  the  activity  of  this  hemolysin  can 
be  made  by  adding  gradually  decreasing  amounts  of  broth  culture 
(filtered  through  unglazed  porcelain)  to  well-washed  red  blood  cells 
suspended  in  salt  solution;  the  mixtures  are  incubated  at  37°  C. 
for  one  hour,  then  kept  in  the  ice  box  twenty-four  hours.  The  greatest 
dilution  of  broth  showing  hemolysis  is  considered  the  unit.1  This 
enzyme  is  destroyed  or  inactivated  at  a  temperature  of  60°  C.  in 
twenty  minutes.  Whether  this  hemolysin  is  identical  with  or  produced 
parallel  to  the  proteolytic  enzyme  of  the  staphylococcus  has  not  been 
determined.  Burckhardt2  believes  the  staphylolysin  is  a  true  hemolytic 
bacterial  toxin;  from  his  observations  it  appears  to  be  non-protein 
in  nature,  not  giving  the  biuret  reaction.  It  is  soluble  in  ether. 

Leucocidin. — Van  de  Velde3  has  obtained  an  enzyme  which  destroys 
leukocytes  by  injecting  virulent  staphylococci  into  the  pleural  cavities 
of  rabbits;  the  exudate,  freed  from  cellular  detritus  by  filtration 
through  unglazed  porcelain,  rapidly  kills  and  even  dissolves  fresh  leuko- 
cytes. Neisser  has  shown  that  fresh  leukocytes  will  reduce  the  color 
of  dilute  methylene  blue  solutions  to  the  point  of  extinction;  if  dilute 
methylene  blue  is  added  to  tubes  containing  leukocytes  and  leuko- 
cidin,  no  reduction  occurs,  thus  indicating  that  the  leukocytes  are  in- 
jured or  destroyed.  Leukocidin  solutions  alone  fail  to  remove  the  color. 

Thrombokinase. — Loeb's  observation4  that  the  products  of  growth 
of  staphylococci  cause  blood  to  coagulate  more  rapidly  than  normal 
.has  been  interpreted  by  Much5  to  be  due  to  a  substance  reacting  like 
a  thrombokinase. 

Distribution  in  Nature. — Staphylococci  are  found  widely  distributed 
in  nature,  but  associated  rather  closely  with  man  and  the  higher 
domestic  animals.  These  organisms  do  not  appear  to  be  adapted  to 
a  purely  saprophytic  existence.  They  are  found  in  dust,  particularly 
that  of  stables,  houses,  and  hospitals;  they  are  common  on  the  skin, 
the  mucous  membranes  of  the  nose,  mouth,  and  to  a  lesser  extent  in 
the  gastro-intestinal  tract,6  the  eye,  the  external  ear,  and  nearly  always 

1  It  must  be  remembered  that  the  sera  of  normal  men  and  of    animals  frequently 
exhibit    antibemolytic    powers,  hence  the  necessity  of   washing  red  blood  cells   thor- 
oughly before  testing  the  activity  of  staphylolysin  upon  them. 

2  Arch,  exp:  Path.  u.  Pharm.,  1910,  Ixiii,  107. 

3  Ann.  Inst.  Past.,  1896. 

4  Jour.  Med.  Res.,  1903,  x,  407. 
6  Biochem.  Zeit.,  1908,  xiv,  143. 

6  Moro,  Jahrb.  f.  Kinderheilk.,  lii,  530;  Streit,  Inaug.  Diss.,  Bonn,  1897. 


260  THE  PYOGENIC  COCCI 

under  the  finger  nails  and  in  the  hair  follicles  in  man,  which  makes 
sterilization  of  the  skin  and  hands  difficult. 

Chemotaxis. — The  bodies  of  staphylococci  appear  to  contain  sub- 
stances of  unknown  composition  which  attract  leukocytes;  the  cell 
substance  of  killed  cocci  injected  in  the  cornea  frequently  causes  an 
accumulation  of  leukocytes  in  the  anterior  chamber  of  the  eye — 
hypopyon. 

Pathogenesis. — Man. — Ordinarily  the  organisms  exist  on  the  intact 
surfaces  of  man  as  "opportunists,"  occasionally  gaining  entrance  to 
the  underlying  tissues  through  abrasions,  chiefly  in  the  skin,  causing 
localized  abscesses,  furuncles,  or  metastatic  inflammations.  Of  the 
metastatic  inflammations,  acute  osteomyelitis  and  endocarditis  are 
the  more  common;  less  commonly  generalized  purulent  pyemias 
develop.  It  is  assumed  that  metastatic  pyemias  are  caused  either  by 
direct  invasion  of  the  blood  stream  or  less  commonly  by  transmission 
of  staphylococci  in  leukocytes  to  remote  parts  of  the  body;  there 
they  escape  from  the  leukocytes  and  set  up  new  foci  of  infection. 
Suppurative  pleurisy  and  pericarditis  are  not  uncommon.  The  occur- 
rence of  furunculosis  in  diabetics  is  so  frequent  as  to  lead  to  the  sup- 
position that  not  only  is  the  general  average  resistance  to  invasion  by 
staphylococci  reduced  in  this  disease,  there  may  be  a  peculiar  local 
lack  of  resistance  in  the  skin  itself.  Occasional  individuals  exhibit 
a  certain  vulnerability  to  infection  in  particular  regions;  the  neck 
and  buttocks  are  more  frequently  affected.  One  invasion  appears  to 
predispose  to  subsequent  infection.  Staphylococci  frequently  are 
secondary  invaders  in  pulmonary  tuberculosis,  diphtheria  and  other 
severe  infections.  Generally  speaking,  staphylococci  cause  acute 
focal  inflammations.  Generalized  infections  of  staphylococcus  causa- 
tion are  relatively  uncommon.  Prolonged  infections  frequently  result 
in  profound  generalized  symptoms;  chills  with  intermittent  fever  are 
the  more  common  clinical  signs.  Parenchymatous  or  even  amyloid 
degeneration  of  certain  glandular  organs,  notably  the  kidneys,  is  the 
more  common  pathological  lesion  in  such  cases. 

Experimental  Reproduction  of  Lesions. — A  satisfactory  explanation 
of  the  pathogenesis  of  staphylococci  for  man  is  not  available.  Neither 
the  staphylolysin  nor  the  leukocidin  appears  to  play  a  prominent  part 
in  the  morbid  process.  There  is  little  definite  evidence  that  the  cell 
substance  of  the  organisms  themselves  is  the  important  factor.  Never- 
theless, the  etiological  relationship  of  staphylococci  to  furunculosis 


THE  STAPHYLOCOCCUS  GROUP  261 

has  been  definitely  established  by  the  experiments  of  Carre1  and  Engels,2 
both  of  whom  rubbed  virulent  cultures  of  these  organisms  upon  their 
skin,  producing  there  typical  furuncles. 

Animals. — Rabbits  are  the  best  of  the  laboratory  animals  for 
experimental  inoculation.  Subcutaneous  inoculations  of  virulent 
strains  frequently  result  in  abscess  formation  and  the  development  of 
a  febrile  reaction.  These  abscesses  commonly  ulcerate,  discharge  and 
heal  spontaneously.  By  no  means  do  all  virulent  strains  induce  lesions, 
however;  there  is  great  difference  between  them  in  this  respect.  Intra- 
peritoneal  injections  frequently  cause  a  rapidly  fatal  peritonitis  with 
or  without  septicemia.  The  intravenous  injection  of  0.25  to  1  c.c. 
of  an  eighteen-hour  broth  culture  usually  causes  a  generalized  pyemia 
with  septic  foci,  particularly  frequent  in  the  kidneys  and  liver.  Orth3 
and  Wyssokowitsch4  have  shown  that  mechanical  injury  to  the  heart 
valves  prior  to  the  intravenous  injection  of  staphylococci  usually 
causes  a  localization  of  the  organisms  there,  producing  an  endocarditis. 
If  a  bone  is  injured  prior  to  an  intravenous  injection,  a  typical  osteo- 
myelitis frequently  results.  It  should  be  remembered  that  the  pus 
produced  by  staphylococci  in  rabbits  is  more  dry  than  that  produced 
in  man.  Guinea-pigs  are  less  susceptible  than  rabbits  to  infection 
with  the  staphylococcus. 

Immunity  and  Immunization. — Staphylococci  do  not  ordinarily 
exhibit  invasive  powers  for  man  or  animals;  they  are  usually  parasitic. 
Whenever  the  continuity  of  the  skin  is  destroyed,  as  by  abrasion,  or 
weakened,  as  in  diabetes,  the  organisms  reach  the  underlying  tissues 
and  induce  inflammatory  reactions.  Repeated  injections  first  of 
killed  then  live  staphylococci  will  frequently  raise  the  threshold  of 
infection  in  experimental  animals  to  a  very  considerable  degree,  but 
the  process  of  immunization  can  not  be  always  relied  upon — many 
animals  die  rather  abruptly  with  rather  extensive  amyloid  degenera- 
tion, particularly  of  the  kidneys.  Leukocytes,  particularly  the  poly- 
morphonuclear  leukocytes,  appear  to  play  a  prominent  part  in  the 
immunity  against  staphylococci;  it  can  be  shown  by  experiment  that 
the  leukocytes  are  more  active  phagocytically  in  immunized  than  in 
non-immunized  animals. 

Similarly,  the  resistance  to  staphylococcus  infection,  which  appears 

1  Fortschritt  d.  Med.,  1885,  170. 

2  Cent.  f.  Bakt.,  Orig.,  1903,  xxxiv,  96. 

3  Cent.  f.  d.  med.  Wissensch.,  1905,  No.  33. 

4  Virchow's  Arch.,  1886,  ciii. 


262  THE  PYOGENIC  COCCI 

to  be  rather  marked  in  the  average  normal  man,  seems  to  depend 
largely  on  the  phagocytic  activity  of  leukocytes  in  the  last  analysis; 
and  the  efficiency  of  vaccines,  particularly  the  autogenous  vaccines, 
in  the  treatment  of  furunculosis  has  focused  attention  sharply  upon 
the  part  played  by  opsonins  in  these  infections.  Generally  speaking, 
injections  of  killed  cultures  of  staphylococci  in  graduated  doses 
beginning  with  one  hundred  millions  and  increasing  to  a  thousand 
millions  or  more  at  appropriate  intervals  exert  a  favorable  influence  on 
the  course  of  the  infection.  The  efficiency  of  this  vaccination  (active 
immunization)  is  attributed  to  the  gradual  development  of  specific 
opsonins  (bacteriotropins)  which  reenforce  the  action  of  normal 
opsonins,  whose  activity  is  somewhat  below  normal.  In  practice 
this  is  accomplished  in  the  following  manner:  the  organism  is  isolated 
on  agar  slants  in  pure  culture,  washed  off,  after  twenty-four  hours' 
incubation,  in  normal  salt  solution,  thoroughly  emulsified,  and  stan- 
dardized so  that  each  cubic  centimeter  contains  the  requisite  number 
of  bacteria.  They  are  killed  either  by  heating  to  80°  C.  for  one  hour, 
or,  better,  by  the  addition  of  Ot5  per  cent,  carbolic  acid,  and  incubation 
at  37°  C.  for  twenty-four  hours.  The  sterility  of  the  culture  must  be 
demonstrated  before  it  is  used.  This  vaccine  is  inoculated  subcutan- 
eiously,  with  surgical  precautions,  using  the  dosage  mentioned  above 
as  a  routine.  The  inoculations  are  repeated  at  intervals  of  from  five 
to  eight  days.  The  duration  of  the  immunity  induced  by  vaccination 
is  not  known.  Vaccines  are  less  effective  in  pyemia  and  metastatic 
staphylococcus  infections  than  in  the  localized  infections. 

The  lessened  lipase  activity  of  the  blood,  manifested  by  a  decreased 
splitting  of  ethyl  butyrate,  is  a  frequent  result  of  staphylococcus 
invasion,  according  to  Clerc;1  according  to  V.  Dungern,2  the  blood 
serum  from  cases  of  extensive  osteomyelitis  is  several  times  as  inhib- 
itory to  the  staphylococcus  enzymes  as  is  that  of  normal  individuals. 

Antibodies. — The  cell  substance  of  staphylococci  does  not  appear 
to  be  very  poisonous  to  experimental  animals,3  and  although  an  anti- 
staphylolysin  and  an  antileukocidin  are  relatively  easily  produced  in 
experimental  animals,  they  do  not  appear  to  confer  any  consider- 
able degree  of  immunity.  Agglutinins  do  not  appear  to  have  been 
demonstrated  in  the  blood  serum  of  man  and  animals  suffering  from 
staphylococcal  infections,  but  Kolb  and  Otto,  and  Proscher4  claim 

1  Compt.  rend.  Soc.  de  biol.,  1901,  liii,  1131. 

2  Munchen.  med.  Wchnschr.,  1898,  xlv,  1040. 

3  Kruse,  Allgemeine  Mikrobiologie,  Leipzig,  1910,  p.  968. 

4  Cent.  f.  Bakt.,  1903,  xxxiv:  quoted  by  Besson,  Practical  Bacteriology,  1913. 


THE  STAPHYLOCOCCUS  GROUP  263 

to  have  prepared  sera  of  marked  agglutinating  value,  which  clump 
virulent  strains  in  higher  dilution  than  non- virulent  strains. 

Precipitins. — Specific  precipitin  reactions  appear  to  have  been 
demonstrated  in  animals  infected  with  staphylococci. 

Bacteriological  Diagnosis. — (a)  Microscopic. — A  Gram  stain  of  the 
suspected  material  usually  suffices  to  establish  a  diagnosis.  It  must 
be  remembered,  however,  that  staphylococci  from  pus  and  exudates 
may  occur  in  pairs  and  even  in  short  chains;  they  may,  therefore, 
be  mistaken  for  streptococci.  An  absolute  diagnosis  can  be  made 
only  by  the  identification  of  pure  cultures. 

(6)  Cultural. — Pure  cultures  of  staphylococci  are  usually  obtained 
readily  by  "streaking  out"  or  plating  the  organisms  on  agar.  Blood 
agar  is  preferable,  if  streptococci  or  pneumococci %  are  also  suspected 


FIG.  33. — Micrococcus  tetragenus.      X  800. 

to  be  present,  otherwise  the  latter  may  be  overlooked.  The  identi- 
fication of  the  colonies  on  agar  usually  can  be  made  by  the  examina- 
tion of  a  Gram-stained  preparation.  Staphylococci  are  common  on 
the  skin,  and  precautions  must  be  taken  to  eliminate  this  source  of 
error  before  making  cultures. 

(c)  Animal  Inoculation. — The  virulence  exhibited  by  staphylococci 
for  animals  is  not  a  reliable  index  of  their  virulence  for  man. 

Dissemination  and  Prophylaxis. — The  wide  distribution  of  staphy- 
lococci on  the  mucous  membranes,  particularly  on  the  skin  and  in 
the  hair  follicles,  makes  the  prevention  of  their  introduction  to  under- 
lying tissues  through  cuts  and  abrasions  difficult.  The  customary 
procedures  of  aseptic  surgery  are  the  best  preventatives  of  infection. 
The  skin  may  be  sterilized  for  operation  (after  thorough  cleansing 
and  drying,  which  is  imperative)  by  painting  with  freshly  prepared 


264  THE  PYOGENIC  COCCI 

tincture  of  iodin  or  iodoform.  Sterilization  is  usually  accomplished 
within  ten  minutes  after  the  iodin  is  applied. 

Staphylococcus  Pyogenes  Citreus. — This  organism  differs  from 
Staphylococcus  aureus  chiefly  in  the  color  of  the  pigment  it  produces, 
a  lemon  yellow,  and  a  lessened  ability  to  liquefy  gelatin. 

Staphylococcus  Pyogenes  Albus. — In  many  instances  this  organism 
is  an  achromogenic  variant  of  Staphylococcus  aureus:  it  produces 
white  colonies  on  agar  and  gelatin,  it  liquefies  gelatin  slowly,  and  it 
is  somewhat  less  pathogenic  for  rabbits;  many  strains  do  not  ferment 
mannite. 

Staphylococcus  Epidermidis  Albus. — Welch  first  described  this 
organism,  which  appears  to  be  a  degenerate  Staphylococcus  albus; 
it  does  not  liquefy  gelatin  and  its  pathogenic  powers  are  practically 
nil.  It  frequently  causes  the  troublesome  but  relatively  benign 

stitch  abscesses."  It  appears  to  be  a  very  constant  parasite  on  the 
skin. 

Micrococcus  Tetragenus. — Micrococcus  tetragenus  was  first  de- 
scribed by  Gaffky;1  he  found  it  in  cavities  of  the  lung  in  pulmonary 
tuberculosis.  It  occurs  but  rarely  in  pure  culture  in  abscesses  either 
in  man  or  animals,2  but  it  is  often  present  in  the  saliva;  occasionally 
it  has  been  recovered  from  dento-alveolar  abscesses.3 

Morphology. — The  organism  occurs  typically  in  tetrads,  enclosed  in 
transparent  gelatinous  capsules  which  require  special  staining  methods 
for  their  demonstration.  The  individual  cells  are  about  1  micron  in 
diameter.  In  artificial  media  the  tetrad  arrangement  may  disappear 
and  the  cocci  occur  chiefly  in  pairs  and  groups  of  three  or  four  pairs. 
The  tetrad  arrangement  and  the  capsule  are  restored  by  passage 
through  animals.  The  organism  is  non-motile,  and  possesses  no  fla- 
gella.  It  forms  no  spores  and  stains  readily  with  ordinary  anilin  dyes. 
It  is  Gram-positive. 

Isolation  and  Culture. — Micrococcus  tetragenus  grows  rather  slowly 
in  all  ordinary  media,  particularly  the  first  transfers  from  the  tissues 
to  artificial  media.  It  can  be  isolated  readily  in  pure  culture  in  gelatin 
or  agar  plates;  the  colonies  are  small,  round  and  grayish,  0.5  to  0.75 
mm.  in  diameter. 

Growth  in  Media. — The  organism  does  not  liquefy  gelatin,  casein, 
or  blood  serum.  Acid  is  produced  in  dextrose,  lactose,  saccharose,  and 

1  Mitt.  a.  d.  kais.  Gesamte,  i,  p.  1. 

2  Miiller,  Wien.  klin.  Wchnschr.,  1904,  xvii,  1815. 

3  Goadby,  Mycology  of  the  Mouth,  1903,  p.  101. 


THE  STAPHYLOCOCCUS  GROUP  265 

mannite  broths.  A  uniform  turbidity  is  produced  in  plain  and  sugar 
broths;  the  growth  is  more  luxuriant  in  the  latter.  Milk  is  slightly 
acidulated,  but  no  coagulation  or  peptonization  takes  place.  Micro- 
coccus  tetragenus  is  aerobic,  facultatively  anaerobic.  The  optimum 
temperature  of  growth  is  37°  C.,  the  maximum  about  44°  C.,  the  mini- 
mum about  12°  C.  The  resistance  to  physical  and  chemical  agents 
is  undetermined. 

Products  of  Growth. — Unknown:  no  toxin  has  been  described. 

Pathogenesis. — The  frequent  occurrence  of  the  organism  in  the 
sputum  of  the  tuberculous  and  its  occasional  isolation  from  tuber- 
culous cavities  has  led  to  the  theory  that  Micrococcus  tetragenus  may 
play  a  secondary  part  in  the  destruction  of  lung  tissue.  This  is  not 
definitely  determined,  however.  It  is  also  found  in  the  saliva  of  healthy 
individuals.  Less  commonly  it  has  been  found  in  the  pus  of  empyemas 
which  follow  pneumonia;  but  the  organism  can  hardly  be  regarded 
as  a  human  pathogen. 

Injected  subcutaneously  into  white  mice,  Micrococcus  tetragenus 
usually  causes  a  fatal  septicemia;  the  organism  may  be  recovered 
from  the  heart  blood,  spleen  and  liver.  House  and  field  mice  appear 
to  be  relatively  immune.  Intraperitoneal  injection  into  guinea-pigs 
may  cause  a  fatal  peritonitis  with  much  pus  in  which  typical  tetrads 
are  found.  Rabbits  and  dogs  are  not  .susceptible.  Infections  with 
the  organism  in  man  are  so  uncommon  that  nothing  is  definitely  known 
of  human  susceptibility  and  immunity.  Vaccines  have  been  tried 
in  a  very  few  cases  with  somewhat  promising  results. 

Bacteriological  Diagnosis. — The  finding  of  Gram-positive  cocci  about 
1  micron  in  diameter  in  pus,  which  occur  habitually  in  tetrads,  usually 
suffices  to  establish  a  satisfactory  bacteriological  diagnosis.  The 
saliva  occasionally  contains  tetracocci  which  resemble  Micrococcus 
tetragenus  very  closely,  but  it  is  claimed  by  many  that  these  organisms 
are  not  necessarily  Micrococcus  tetragenus,  Isolation  and  identifica- 
tion by  cultural  methods  must  be  resorted  to  in  suspected  cases. 

Micrococcus  Ovalis. — Synonym. — Enterococcus.1 

Historical. — Micrococcus  ovalis  was  described  by  Escherich,2  who 
found  it  very  commonly  in  the  intestinal  tracts  of  nurslings  and  bottle- 
fed  infants. 

Morphology. — The  organism  is  oval  in  outline,  measuring  0.6  to 
0.9  microns  in  the  lesser  diameter,  and  it  occurs  habitually  in  pairs, 

1  Thiercelin,  Th&se  de  Paris,  1894. 

2  Darmbakterien  des  Sauglings,  Stuttgart,  1886,  p.  89. 


266  THE  PYOGENIC  COCCI 

with  a  tendency  for  the  proximal  ends  to  be  slightly  flattened  and  the 
distal  ends  to  be  somewhat  pointed.  In  this  respect  Micrococcus 
ovalis  resembles  the  pneumococcus  very  closely.  In  fluid  media,  par- 
ticularly sugar  broths,  the  pairs  of  organisms  remain  adherent  in  chains 
of  greater  or  lesser  length  giving  rise  to  a  diplostreptococcus  arrange- 
ment which  is  precisely  like  that  exhibited  by  the  pneumococcus  under 
the  same  conditions. 

Micrococcus  ovalis  is  non-motile  and  possesses  no  flagella.  It  forms 
no  spores.  According  to  Lewkowicz1  and  others,  capsules  are  produced 
when  the  organism  is  isolated  directly  from  lesions.  The  organism 
stains  readily  with  ordinary  anilin  dyes,  and  it  is  Gram  positive. 

Isolation  and  Culture. — Micrococcus  ovalis  grows  with  moderate  vigor 
on  agar  plates,  better  in  dextrose  or  lactose  agar.  The  colonies  after 
forty-eight  hours'  incubation  at  37°  C.  are  round,  translucent,  color- 
less, and  measure  about  1  to  2.5  microns  in  diameter.  They  are  not 
distinctive.  Colonies  on  gelatin  plates  are  very  small  and  develop 
slowly.  The  medium  is  not  liquefied.  Blood  agar  appears  to  be  a 
better  medium  for  isolation  of  Micrococcus  ovalis  than  any  other; 
the  colonies  are  1  to  3  mm.  in  diameter  even  after  eighteen  hours' 
incubation,  grayish  and  succulent.  No  hemolysis  takes  place.  A 
slight  turbidity,  which  soon  settles,  forms  in  plain  broth;  the  addition 
of  dextrose  or  lactose  greatly  enriches  the  growth.  Milk  is  usually 
coagulated  in  one  to  three  days  (acid  coagulation),  but  the  coagulum 
does  not  become  digested. 

Micrococcus  ovalis  is  an  aerobic,  facultatively  anaerobic  organism. 
The  lower  limit  of  growth  is  about  8°  C.,  the  optimum  from  37°  to 
39°  C.,  and  the  maximum  about  45°  C.  Its  resistance  to  chemical 
and  physical  agents  is  about  the  same  as  that  of  the  streptococcus. 

Products  of  Growth. — Chemical. — The  organism  exhibits  no  evidence 
of  proteolysins ;  it  is  relatively  inert  in  protein  media.  No  indol, 
skatol  or  volatile  sulphur  compounds  are  produced.  Acid  is  produced 
in  dextrose  and  lactose  broths;  the  action  on  other  sugars  is  yet  to 
be  determined. 

Enzymes. — No  enzymes  are  known. 

Toxins. — No  toxic  products  have  been  detected  in  cultures  of 
Micrococcus  ovalis. 

Distribution. — The  normal  habitat  of  Micrococcus  ovalis  appears 

to  be  the  intestinal  tract  of  man;  it  occurs  in  the  meconium  frequently,2 

i 

1  Cent.  f.  Bakt.,  1901,  xxix,  635. 

2  Escherich,  loc.  cit. 


THE  STAPHYLOCOCCUS  GROUP  267 

and  it  is  a  constant  inhabitant  of  the  intestinal  flora  of  artificially  fed 
infants;  it  also  occurs  commonly,  but  in  lesser  numbers,  in  the  intes- 
tinal flora  of  the  normal  nursling.  The  organism  has  been  repeatedly 
isolated  from  the  feces  of  adults,  and  it  has  also  been  isolated  from  the 
intestinal  tract  of  cattle.1 

Pathogenesis. — Man. — Micrococcus  ovalis  is  ordinarily  a  harmless 
parasite  of  the  intestinal  tract;  occasionally  it  becomes  invasive 
(usually  secondarily)  and  produces  various  inflammations,  according 
to  the  tissues  invaded  and  its  association  with  other  bacteria.  Lewko- 
wicz2  isolated  Micrococcus  ovalis  in  nearly  pure  culture  from  three 
cases  of  severe  dysentery;  the  organisms  were  found  to  be  capsulated 
and  resembled  pneumococci  in  a  striking  manner.  Jouhaud,3  Thier- 
celin,4  Ramonovitsch,5  and  Gilbert  and  Lippman6  have  isolated  the 
organism  either  in  pure  culture  or  in  association  with  other  bacteria 
from  cases  of  cholecystitis,  puerperal  fever,  appendicitis,  various 
intestinal  inflammations,  and  even  from  the  cerebrospinal  canal  in 
cases  of  meningitis.  The  close  resemblance  of  the  organism  to  the 
pneumococcus,  which  has  been  observed  by  Kruse,7  Sittler8  and  others, 
has  doubtless  led  to  confusion;  many  cases  of  "pneumococcus"  infec- 
tion of  the  stomach,  gall-bladder,  appendix  and  other  intestinal  adnexa 
are  probably  infections  with  Micrococcus  ovalis,  and  vice  versa. 

Animal. — Wilhelmi9  has  isolated  Micrococcus  ovalis  from  enteritides 
of  young  cattle;  Lewkowicz10  has  found  the  organism  isolated  directly 
from  human  inflammations  to  be  pathogenic  for  white  mice.  It  exhibits 
no  pathogenicity  as  it  occurs  normally  in  the  intestinal  tract.11 

Bacteriological  Diagnosis. — 1.  Microscopical. — The  presence  of  con- 
siderable numbers  of  diplococci  in  the  feces  with  their  approximated 
ends  slightly  flattened,  their  distal  ends  somewhat  pointed,  staining 
intensely  with  the  Gram  stain,  is  frequently  sufficient  evidence  to 
establish  a  tentative  diagnosis  of  Micrococcus  ovalis. 

2.  Cultural. — Various  dilutions  of  feces  or  products  of  inflammation 
are  plated  either  on  dextrose  agar  or  "streaked  out"  on  blood  agar. 

1  Wilhelmi,  Landwirthschaft.  Jahrb.  f.  Schweiz.,  1899,  xiii. 

2  Cent.  f.  Bakt.,  1901,  xxix,  635. 

3  These  de  Paris,  1903. 

4  Comp.  rend.  Soc.  de  biol.,  1902,  No.  27;  1908,  Ixiv,  76. 
6  Ibid.,  1911,  Ixx,  122. 

6  Ibid.,  1902,  No.  30. 

7  Cent.  f.  Bakt.,  Orig.,  1903,  xxxiv,  737. 

8  Die  wichtigsten  Bakterientypen  der  Darmflora  beim  Sauglinge,  u.  s.  w.,  Wurzburg, 
1909. 

9  Landwirthschaftl.  Jahrb.  f.  Schweiz.,  1899,  xiii. 

10  Loc.  cit. 

11  Thiercelin,  These  de  Paris,  1894;  Compt.  rend.  Soc.  de  biol.,  April  15,  1899.    Jou- 
haud, These  de  Paris,  1903. 


268  THE  PYOGENIC  COCCI 

The  morphology  and  cultural  reactions  outlined  above  suffice  to  estab- 
lish a  diagnosis.  The  absence  of  hemolysis  or  of  green  discoloration 
of  the  hemoglobin  separates  the  streptococcus  and  pneumococcus 
from  Micrococcus  ovalis. 

3.  Serological. — Not  practicable. 

Dissemination  and  Prophylaxis. — Micrococcus  ovalis  does  not  cause 
progressive  disease  from  man  to  man;  it  is  an  intestinal  parasite 
habitually  and  only  occasionally  becomes  invasive.  No  precautions 
other  than  the  careful  sterilization  of  dejecta  are  necessary.  The 
hands  of  attendants  should  be  kept  surgically  clean  when  caring  for 
intestinal  disturbances  incited  by  Micrococcus  ovalis,  or,  indeed,  by 
any  microorganism. 


CHAPTER  XIII. 
THE  STREPTOCOCCUS-PNEUMOCOCCUS  GROUP. 


THE  STREPTOCOCCUS  GROUP. 
Streptococcus  Pyogenes. 


Streptococcus  Einheit  or  Vielheit. 
THE  PNEUMOCOCCUS. 


THE   STREPTOCOCCUS   GROUP. 

THE  Streptococcus  Group  comprises  those  spherical  bacteria  in 
which  as  multiplication  proceeds  the  successive  planes  of  division  are 
parallel  and  the  individual  cells  remain  adherent  in  longer  or  shorter 
chains.  The  limits  of  the  group  are  poorly  defined,  both  morphologi- 
cally and  pathogenically.  It  includes  organisms  which  occur  habitually 
in  chains,  both  in  culture  and  in  the  animal  body,  and  its  limits  have 
been  extended  to  enclose  types  which  exhibit  chain  formation  only 
in  fluid  media.  The  latter,  of  which  Micrococcus  ovalis  and  the 
pneumococcus  are  examples,  occur  in  the  animal  body  as  diplo^^i, 
and  grow  thus  on  solid  media;  in  fluid  media  they  grow  habitually 
in  chains  of  greater  or  lesser  length,  in  which,  however,  the  typical 
diplococcal  arrangement  persists.  The  term  streptococcus,  there- 
fore, is  a  purely  morphological  one;  it  includes  organisms  which  excite 
various  types  of  inflammation  in  man  and  in  animals,  together  with 
those  which  are  ordinarily  saprophytic. 

The  most  important  members  of  the  group  exist  on  the  skin,  and 
particularly  on  the  mucous  membranes  of  man,  as  habitual  parasites 
or  "opportunists."  Streptococcus  pyogenes  and  its  variants  are  the 
most  common  of  these  and  the  most  versatile  in  their  pathogenesis. 

Streptococcus  Pyogenes. — Synonyms. — Streptococcus  erysipelatos ; 
Streptococcus  scarlatinosus;  Streptococcus  septicus. 

Historical. — Streptococci  were  seen  in  unstained  pus  by  Klebs  in 
1872.  Several  years  later  Koch1  demonstrated  them  in  stained  sec- 
tions and  in  inflammatory  exudates.  Pasteur2  appears  to  have  been 
the  first  to  cultivate  streptococci  from  cases  of  puerperal  fever  and  to 
differentiate  them  from  staphylococci,  both  morphologically  and  by 

1  Untersuchungen  liber  Wundinfektion,  1878. 

2  Compt.  rend.  Acad.  sci.,  1880,  xc,  1035. 


270  STREPTOCOCCUS-PNEUMOCOCCUS  GROUP 

the  character  of  the  lesions  which  they  excite.  Ogsten1  independently 
confirmed  Pasteur's  observations.  Fehleisen,2  using  more  exact  cul- 
tural methods,  isolated  streptococci  from  a  case  of  erysipelas;  Rosen- 
bach3  studied  the  organism  in  great  detail  and  introduced  the  name, 
Streptococcus  pyogenes. 

Morphology. — The  individual  cells  are  spherical,  less  commonly 
oval,  measuring  from  0.5  to  1  micron  in  diameter.  The  size  of 
individual  cells  varies  somewhat  even  in  the  same  culture.  The 
organisms  remain  adherent  in  chains  which  vary  in  length  from  four 
to  twenty  or  more  elements,  in  which  a  definite  association  of  cocci 
in  pairs  with  their  proximate  sides  flattened  is  occasionally  observed. 
The  number  of  elements  in  the  chain  varies  somewhat  according  to  the 
origin  of  the  culture;  it  has  been  observed  that  streptococci  freshly 
isolated  from  lesions  tend  to  occur  in  longer  chains,  while  those  organ- 
isms which  grow  habitually  upon  the  normal  surfaces  and  mucous 
membranes  of  the  body  appear  more  frequently  in  shorter  chains. 
V.  Lingelsheim4  has  designated  those  strains  which  form  chains  of 
eight  or  more  cocci,  Streptococcus  longus;  the  short-chain  types  are 
called  Streptococcus  brevis.  Notwithstanding  the  frequent  parallel- 
ism of  pathogenesis  and  development  of  long  chains  of  cocci  in  artificial 
media,  in  contradistinction  to  the  lesser  virulence  of  the  short-chain 
types,  experience  has  shown  that  the  length  of  the  chains  may  also 
be  influenced  directly  by  variations  in  the  culture  media.5  This  dis- 
tinction, therefore,  is  untenable.  Streptococci  grown  on  solid  media 
are  prone  to  group  themselves  in  pairs,  or  even  irregular  masses, 
resembling  staphylococci.  Similarly,  the  typical  streptococcal  arrange- 
ment is  frequently  lacking  in  purulent  inflammations  of  streptococcal 
causation.  Occasional  cells  in  a  chain  of  streptococci,  especially  in 
old  cultures,  are  met  with  which  are  distinctly  larger  than  their  fellows ; 
they  color  somewhat  differently  and  were  formerly  regarded  as  spores 
— arthrospores.6  It  is  now  known  that  they  are  not  noticeably  more 
resistant  than  the  more  typical  cells,  and  they  are  probably  to  be 
regarded  as  involution  forms. 

Streptococcus  pyogenes  is  non-motile,  non-flagellated,  and  does 
not  produce  true  endospores.  Occasional  strains,  isolated  directly 

1  Brit.  Med.  Jour.,  1881. 

2  Aetiol.  d.  Erysipelas,  Berlin,  1883. 

3  Mikroorganismen  bei  Wundinfektions-Krankh.  des  Menschen,  Wiesbaden,  1884. 

4  Zeit.  f.  Hyg.,  1891,  x,  331. 

5  Hueppe,  Die  Methoden  der  Bakterien-Forschung,  Wiesbaden,  1889,  24,  130. 

6  See  Aronson  (Berl.  klin.  Wchnschr.,  1896,  No.  32;  1902,  No.  42)  and  Vincent  (Arch, 
de  med.  exp.,-etc.,  1902)  for  details. 


THE  STREPTOCOCCUS  GROUP  271 

from  lesions  or  from  animals,  exhibit  a  delicate  stainable  zone  around 
individual  organisms  or  pairs  of  organisms,  which  suggests  capsules. 
Howard  and  Perkins1  have  isolated  such  an  organism  which  exhibited 
a  very  definite  capsule.  It  grew  habitually  in  short  chains  in  fluid 
media,  the  individuals  occurring  typically  in  pairs.  The  organism 
is  closely  related  to  the  pneumococcus,  and  Dochez  and  Gillespie2 
have  named  it  Pneumococcus  mucosus. 

Streptococcus  pyogenes  stains  readily  with  ordinary  anilin  dyes. 
It  is  typically  Gram-positive,  although  old  cultures  may  fail  to  retain 
the  Gram  stain.  The  saprophytic  types  frequently  are  Gram-negative. 

Isolation  and  Culture. — Streptococci  may  be  isolated  directly  from 
inflamed  areas  and  from  pus  upon  agar  plates,  better  upon  dextrose 
agar  plates.  The  colonies  are  minute,  gray  and  transparent,  and 
may  be  readily  overlooked;  if  they  occur  in  association  with  staphy- 
lococci  or  other  rapidly  growing  organisms,  they  are  readily  over- 
grown. The  more  virulent  varieties  develop  less  readily,  and  require 
the  addition  of  blood  or  ascitic  fluid  to  ordinary  media  for  their  initial 
growth  outside  the  body.  On  blood  agar  plates  (one  part  human  blood, 
two  parts  of  nutrient,  sugar-free  agar)  the  majority  of  virulent  strep- 
tococci produce  a  wide,  clear  zone  of  hemolysis  4  to  8  mm.  in  diameter 
around  each  colony.  This  medium  is  particularly  valuable  for  the 
isolation  of  streptococci.3  On  Loffler's  blood  serum  growth  is  mod- 
erately luxuriant;  typical  chains  are  found  in  the  condensation  water 
of  solid  media,  but  not  as  a  rule  upon  the  surface.  The  organisms 
grow  feebly  in  gelatin  stab  cultures  producing  a  few  small  discrete 
gray  colonies  along  the  line  of  inoculation.  Little  or  no  growth  is 
found  on  the  surface  of  the  medium.  Liquefaction  does  not  take  place. 

A  slightly  alkaline  reaction  (neutral  to  phenolphthalein)  is  most 
favorable  for  the  growth  of  streptococci;  the  addition  of  sugars,  par- 
ticularly dextrose,  to  ordinary  media  (but  not  blood  agar)  increases 
the  rate  and  extent  of  development,  which,  however,  are  soon  limited 
by  the  accumulation  of  acid  products  of  fermentation.  The  addition 

1  Jour.  Med.  Research,  1901,  vi,  163. 

2  Jour.  Am.  Med.  Assn.,  1913,  Ixi,  727. 

3  Schottmuller  (Munch,  med.  Wchnschr.,  1903,  xx,  849)  has  classified  streptococci 
according  to  the  changes  they  produce  in  blood  agar  as  follows: 

I.  Streptococcus  longus  pyogenes  seu  erysipelatis  (Streptococcus  pyogenes)  produces  a 
wide,  clear  zone  of  hemolysis  around  the  colony;  in  blood  broth  the  color  changes  to  a 
burgundy  red.    Long-chained  streptococci. 

II.  Streptococcus  mitior  seu  viridans  (Streptococcus  viridans)  produces  a  greenish  area 
around  the  colony;  a  brownish  color  in  blood  broth.    Short-chained  streptococci. 

III.  Streptococcus  mucosus.   No  hemolysis  on  blood  agar.   Colonies  viscid.   Organisms 
distinctly  encapsulated. 


272  STREPTOCOCCUS-PNEUMOCOCCUS  GROUP 

of  solid  calcium  carbonate  (marble)  to  sugar  media  is  important; 
it  neutralizes  the  excess  of  acid,  and  also  appears  to  add  somewhat  to 
the  nutritive  value  of  the  medium.1 

Streptococci  grow  slowly  in  plain  broth,  producing  a  sediment  after 
twenty-four  to  forty-eight  hours'  incubation.  A  flocculent  sediment 
consisting  of  long  chains  of  organisms  is  characteristic  but  not  distinc- 
tive of  many  virulent  strains  (Streptococcus  conglomeratus) ;  a 
granular  sediment  usually  contains  short-chain  streptococci  almost 
exclusively. 

Streptococcus  pyogenes  ferments  dextrose,  lactose,  maltose  and 
saccharose  and  sorbite  with  the  formation  of  considerable  amounts  of 
acid.  Mannite  is  not  as  a  rule  attacked.  Milk  is  coagulated  in  from 
three  to  five  days,  the  coagulum  resulting  from  the  accumulation  of 
the  acid  fermentation  of  the  lactose.  The  coagulum  is  never  dissolved. 
Andrewes  and  Horder2  state  that  Streptococcus  pyogenes  does  not 
coagulate  milk,  although  the  organism  produces  a  considerable  amount 
of  acid  in  this  medium.  Smith  and  Brown3  have  shown  that  boiling 
the  milk  may  be  necessary  to  make  the  coagulum  visible. 

Streptococcus  pyogenes  is  an  aerobic,  facultatively  anaerobic 
organism.  Pathogenic  strains  do  not  as  a  rule  grow  below  16  to  18° 
C.  The  optimum  temperature  lies  between  35°  and  39°  C.,  the  maxi- 
mum about  44°  C.  The  parasitic  types  are  not  long-lived  away  from 
the  human  body.  Exposure  to  60°  C.  for  one  hour  will  kill  most 
streptococci;  a  longer  time  is  required  if  the  organisms  are  exposed 
in  albuminous  media.  Five  per  cent,  carbolic  acid  and  1  to  1000  mer- 
curic chloride  will  kill  the  naked  germs  in  from  five  to  ten  minutes. 
Streptococci  dried  in  sputum  will  resist  a  temperature  of  100°  C. 
(in  flowing  steam)  for  several  minutes,  and  drying  at  ordinary  tem- 
peratures in  the  dark  for  several  weeks.  Direct  sunlight  kills  them  in 
about  ten  hours.  The  organisms  survive  and  retain  their  virulence 
if  they  are  suspended  in  sterile,  defibrinated  blood  and  kept  in  the 
ice  box  for  several  weeks. 

Products  of  Growth. — Chemical. — Streptococci  exhibit  but  little 
evidence  of  proteolytic  activity.  No  indol,  skatol,  phenol  or  other 
aromatic  derivatives  of  amino  acids  have  been  detected  in  cultures; 
gelatin  is  not  liquefied  and  casein  and  coagulated  blood  serum  are 
not  visibly  changed.  Emmerling4  found  peptone,  leucin,  ty rosin, 

1  Bolduan,  New  York  Med.  Jour.,  1905,  May  13. 

2  Lancet,  1906,  ii,  708. 

3  Jour.  Med.  Research,  1914,  xxxi,  455. 
*  Berl.  chem.  Gesell.,  1897,  1863. 


THE  STREPTOCOCCUS  GROUP  273 

ammonia,  methylamine,  propyl  pyridin,  succinic  acid,  butyric  acid 
and  other  volatile  acids  among  the  anaerobic  decomposition  products 
of  fibrin  by  this  organism,  but  no  aromatic  derivatives. 

Toxin. — A  soluble  toxin  has  not  been  demonstrated  in  cultures  of 
streptococci,  although  substances  have  been  isolated  by  Marmorek1 
and  others  which  will  kill  guinea-pigs.  These  substances  do  not 
exhibit  sufficient  potency  to  warrant  the  assumption  that  they  are 
important  factors  in  the  production  of  the  grave  symptoms  charac- 
teristic of  severe  streptococcus  infections.  Attempts  to  demonstrate 
endotoxin  have  also  been  unsuccessful;  the  bodies  of  the  organisms 
are  but  slightly  toxic  to  experimental  animals.  The  manifestations 
of  toxemia  in  streptococcal  infections,  however,  are  too  striking  to 


FIG.  34. — Streptococcus  in  pus.    X  800. 

be  reconciled  with  the  negative  results  of  these  investigations;  the 
nature  of  the  mechanism  of  streptococcus  infection  remains  to  be 
elucidated. 

Hemolysin — Streptocolysin. — Bordet2  and  Besredka3  have  shown 
that  filtered  broth  cultures  of  streptococci  will  dissolve  red  blood 
corpuscles,  liberating  hemoglobin,  and  that  this  hemolytic  substance 
— streptocolysin — is  active  both  in  mw  and  in  vitro.  Frequently  the 
blood  of  rabbits  injected  with  streptocolysin  was  found  to  be  ulaked" 
just  before  death.  Besredka's  observations  would  indicate  that  the 
substance  is  rather  firmly  bound  to  the  organisms  and  does  not  appear 
in  the  medium  to  any  considerable  degree.  M'Leod,4  M'Leod  and 

1  Berl.  klin.  Wchnschr.,  1902,  xiv,  253. 

2  Ann.  Inst.  Past.,  1897,  xi,  177. 
a  Ibid.,  1901,  xv,  880. 

4  Jour.  Path,  and  Bact.,  1912,  xvi,  321. 

18 


274  STREPTOCOCCUS-PNEUMOCOCCUS  GROUP 

M'Nee,1  and  Lyall2  have  studied  the  conditions  favoring  the  formation 
of  the  hemolysin  and  find  that  sugar-free  ascitic  broth  is  suitable  for 
this  purpose.  The  substance  is  thermolabile  and  is  found  in  an  active 
state  only  during  the  first  twelve  to  twenty-four  hours  of  culture, 
at  which  time  small  amounts  of  sterile  (filtered)  broth,  0.01  to  0.10 
c.c.,  are  strongly  hemolytic.  The  hemolysin  does  not  induce  antibody] 
formation  when  it  is  injected  into  susceptible  animals.  Hemoglobin- 
emia  and  hemoglobinurea  are  produced  in  rabbits  that  are  very  sus- 
ceptible to  the  hemolysin;  less  susceptible  rabbits-  react  but  slightly. 
There  is  no  definite  evidence  that  streptocolysin  plays  a  prominent 
part  in  the  streptococcus  infections  of  man.  Virulence  and  hemolytic 
activity  are  frequently,  but  by  no  means  necessarily,  parallel  pheno- 
mena. 

Distribution  in  Nature. — Streptococci  are  widely  distributed  in  nature, 
always,  however,  in  rather  intimate  association  with  man  or  the 
higher  animals.  They  are  found  in  the  soil,  water,  milk,  and  they 
exist  as  "opportunists"  on  the  exposed  surfaces  and  mucous  mem- 
branes of  man.  They  are  common  in  the  mouth,  nose  and  throat,  the 
intestinal  tract,  and  rare  in  the  normal  vagina. 

Pathogenesis. —  Human. — Streptococci  excite  both  local  inflam- 
matory and  suppurative  processes  and  generalized  septicemic  infec- 
tions, the  latter  being  the  more  common  and  characteristic.  Super- 
ficial lesions  may  be  mild  in  character,  resembling  those  caused  by 
staphylococci.  The  organisms  may,  and  frequently  do,  enter  the 
blood  or  lymph  channels,  and  spread  rapidly  through  the  body,  incit- 
ing the  most  severe  generalized  infections.  Streptococci  are  the  etio- 
logical  agents  of  erysipelas,  frequently  of  general  and  puerperal  sepsis 
and  phlebitis,  and  inflammations  of  the  internal  organs;  of  these, 
the  middle  ear,  the  endocardium,  the  peritoneum,  the.meninges  or 
joints  are  more  commonly  involved.3  Escherich4  and  others  have 
described  a  severe  type  of  enteritis,  particularly  of  young  children — 
streptococcus  enteritis — which  occasionally  exhibits  an  epidemic 
tendency  in  the  summer  months.5  Attention  has  been  directed  in 
recent  years  to  severe  epidemics  of  septic  sore  throat  in  which  the 

1  Ibid.,  1913,  xvii,  524. 

2  Jour.  Med.  Research,  1914,  xxx,  487. 

3  Menzer,  Deut.  med.  Wchnschr.,  1901,  97.     Meyer,  Zeit.  f.  klin.  Med.,  1902,  xlvi, 
311;    Internal.  Beitrage  zur  inn.  Med.,   1902,  ii,  443.     Philipp,   Deut.  Arch.  f.   klin. 
Med.,  1903,  Ixxvi,  150.    Poynton  and  Payne,  Cent.  f.  Bakt.,  Orig.,  1902,  xxxi,  502.    Cole, 
Jour.  Inf.  Dis.,  1904,  i,  714.    Rosenow,  Jour.  Inf.  Dis.,  1910,  vii,  411;    ibid.,  1912,  xi, 
210;  Jour.  Am.  Med.  Assn.,  1913,  Ix,  1223. 

4  Jahrb.  f.  Kinderheilk.,  1899,  xlix,  137. 

5  Kendall,  Day  and  Bagg,  Boston  Med.  and  Surg.  Jour.,  1913,  clxix,  741. 


THE  STREPTOCOCCUS  GROUP  275 

evidence  points  to  streptococci  transmitted  through  milk  as  the 
etiological  agent.  The  type  of  streptococcus  involved  has  been  a 
subject  of  controversy,  but  the  extensive  studies  of  Smith  and  Brown1 
show  clearly  that  Streptococcus  pyogenes  is  by  far  the  most  common 
organism  found.  They  demonstrated  that  the  streptococcus  which 
is  isolated  from  bovine  mastitis  is  not,  except  possibly  in  rare  instances, 
a  causative  factor  in  epidemic  sore  throat. 

Streptococci  occur  frequently  as  secondary  invaders  in  diphtheria, 
many  gastro-intestinal  diseases,  and  diseases  of  the  lungs,  where  they 
may  be  at  times  even  more  formidable  than  the  primary  infecting 
organism.  As  Theobald  Smith  has  admirably  expressed  it,  they  are 
''organisms  of  the  diseased  state."  The  virulence  exhibited  by  strep- 
tococci varies  considerably,  as  does  the  type  of  lesions  they  excite.  This 
variation  in  virulence  is  not  at  all  well  understood  at  the  present  time, 
but  experiments  indicate  that  the  site  of  infection  and  the  past  history 
of  the  organism  exercise  some  influence.  Rosenow2  has  isolated 
streptococci,  using  special  methods,  from  the  regional  glands  in  arth- 
ritis, gall-bladders,  and  gastric  ulcers.  He  states  that  the  freshly- 
isolated  strains  exhibit  rather  marked  tendencies  to  localize  in  the 
homologous  tissues  of  experimental  animals.  This  specific  tissue 
affinity  is  rapidly  lost  during  cultivation  of  the  organisms  in  artificial 
media,  however. 

Animal. — Frankel,3  Petruschky,4  and  Koch  and  Petruschky5  showed 
that  the  virulence  of  the  same  strain  of  streptococcus  varied  materially 
according  to  the  conditions  of  culture,  and  that  the  lesions  produced 
in  rabbits  varied  likewise;  thus  the  descendants  of  the  same  culture 
would  produce  variously  a  rapidly  fatal  septicemia,  erysipelas,  arth- 
ritis, endocarditis  or  peritonitis.  Marmorek  has  shown  that  the  viru- 
lence of  streptococci  for  animals  may  be  greatly  increased  by  repeated 
passage;  after  a  series  of  passages  an  incredibly  small  amount  of  cul- 
ture, even  one  one-hundred-millionth  of  a  cubic  centimeter  of  a  forty- 
eight-hour  broth  culture  introduced  intraperitoneally  may  cause  death 
within  two  days.  Streptococci  which  are  virulent  for  man  frequently 
exhibit  but  little  virulence  for  animals;  it  is  essential,  therefore, 
that  large  amounts  of  material  be  injected  into  experimental  animals 
to  obtain  infection.  Rabbits  are  more  susceptible  than  other  labora- 

Jour.  Med.  Research,  1914,  xxxi,  455. 

Jour.  Am.  Med.  Assn.,  1913,  Ix,  1223;  Ixi,  1947;  1914,  Ixiii,  1835.  Jour.  Inf.  Dis., 
1915,  xvi,  No.  2. 

Cent.  f.  Bakt.,  1889,  vi,  671. 
Zeit.  f.  Hyg.,  1896,  xxiii,  144. 
Ibid.,  p.  478. 


276 


STREPTOCOCC US-PNE UMOCOCC US  GRO UP 


tory  animals.  Subcutaneous  injections  of  morbid  material  into 
rabbits  result  variously,  depending  upon  the  virulence  of  the  strain 
for  this  animal  (not  necessarily  upon  its  virulence  for  man) ;  a  localized 
abscess  may  form  or  an  erysipelatoid  inflammation  may  occur,  which 
is  usually  somewhat  localized,  but  may  develop  into  a  wide-spread 
cellulitis.  Intraperitoneal  injections  are  usually  followed  by  rapidly- 
fatal  peritonitis.  Death  may  occur  within  twenty-four  hours.  Intra- 
venous injections  may  cause  a  rapidly  fatal  generalized  septicemia,  or, 
if  the  strain  is  less  virulent  and  death  does  not  occur  during  the  first 
three  to  four  days,  the  serous  surfaces  may  be  violently  inflamed.  Less 
virulent  strains  which  do  not  cause  acute  death  usually  lead  to  endo- 
cardial  or  joint  involvement.  Mice  are  nearly  as  susceptible  to  strepto- 


FIG.  35. — Streptococci  in  liver,  section  stained  by  Gram's  method.     X  800.    (KoJle  and 

Hetsch.) 

• 

coccus  infection  as  rabbits.  Guinea-pigs  are  less  susceptible;  subcu- 
taneous inoculations  usually  lead  to  abscess  formation,  which  soon 
heals,  but  intraperitoneal  injections  may  result  in  peritonitis  and 
death.  Horses  are  quite  susceptible  to  infection  with  streptococci, 
particularly  with  Streptococcus  equi  (Streptococcus  coryzse  contagiosse 
equorum),  which  causes  equine  distemper  or  strangles.  The  udders  of 
milch  cattle  occasionally  become  infected  with  streptococci  result- 
ing in  a  severe  inflammation,  mastitis  or  garget,  which  may  lead  to 
loss  of  function  of  one  or  more  quarters  of  the  udder.  It  is  probable 
from  the  investigations  of  Smith  and  Brown1  that  streptococci  of 
bovine  origin  are  not  commonly  the  etiological  agents  of  septic  sore 
throat  in  man. 

1  Loc.  cit. 


THE  STREPTOCOCCUS  GROUP  277 

Immunity  and  Immunization. — Streptococcus  infections,  mild  or 
severe,  do  not  appear  to  induce  any  considerable  degree  of  active 
immunity.  Not  infrequently  recovery  is  a  matter  of  some  time;  the 
acute  symptoms  may  abate  and  the  organisms  disappear  from  the 
blood  stream,  only  to  localize  in  some  internal  organ,  a  structure  as  for 
example,  a  joint,  where  they  may  cause  a  chronic,  obstinate  arthritis. 
It  is  possible  that  various  strains  of  streptococci  which  can  not  be 
differentiated  by  our  somewhat  artificial  cultural  criteria  may  exist, 
and  that  subsequent  infection  may  be  with  another  strain.  A  similar 
condition  exists  in  lobar  pneumonia.  Van  de  Velde1  has  stated  that 
the  serum  of  an  animal  immunized  against  one  strain  of  streptococcus 
will  protect  against  the  homologous  strain,  but  not  against  hetero- 
logous  strains  of  streptococci,  a  somewhat  parallel  situation.  On  the 
other  hand,  experiments  are  recorded  which  are  not  in  accord  with 
this  hypothesis.  A  patient  suffering  from  an  inoperable  tumor  was 
inoculated  subcutaneously  with  a  culture  of  streptococcus;  the 
inoculation  resulted  in  a  moderately  severe  erysipelas  which  per- 
sisted for  about  ten  days;  when  the  inflammation  had  subsided  a 
second  reinoculation  was  made  in  the  same  place,  and  a  secondary 
erysipelatoid  inflammation  spread  over  the  same  area.  A  third 
inoculation  resulted  similarly.  These  experiments  indicate  that  this 
patient  did  not  develop  immunity  at  the  site  of  infection.2 

Rabbits  have  been  actively  immunized  to  streptococci  through 
repeated  vaccination,  first  with  killed  cultures,  then  gradually 
increasing  doses  of  living,  virulent  organisms;  eventually  the  animals 
will  resist  successfully  several  times  the  original  fatal  dose  of  the 
homologous  strain.  Active  immunization  with  polyvalent  vaccines 
containing  many  strains  of  streptococci  from  lesions  is  considerably 
more  efficient  in  protecting  the  animal  against  subsequent  infection 
with  heterogeneous  strains.  The  sera  of  such  actively  immunized 
animals  do  not  possess  noteworthy  antihemolytic  properties;  their  anti- 
toxic content,  if  indeed  there  be  any,  is  unknown.  The  chief  demon- 
strable change  in  the  serum  appears  to  be  an  increased  phagocytic 
power,  causing  Jeukocytes  in  vitro  to  take  up  more  streptococci  than 
they  would  normally.  The  injection  of  sera  of  actively  immunized 
animals  appears  to  increase  the  resistance  of  non-immunized  animals 
to  otherwise  fatal  amounts  of  streptococci. 

1  Cent.  f.  Bakt,,  1898,  xxiv,  688. 

2  Coley  has  injected  streptococci  into  malignant  tumors  with  occasional    beneficial 
results;  the  observations  are  too  few  to  warrant  any  definite  statement  of  the  efficiency 
of  the  procedure. 


278  STREPTOCOCCUS-PNEUMOCOCCUS  GROUP 

Marmorek,1  Tavel2  and  others  have  prepared  antistreptococcic 
immune  sera  on  a  large  scale  by  immunizing  horses  first  with  killed 
cultures,  then  with  increasing  amounts  of  living  cultures.  Marmorek, 
a  staunch  supporter  of  the  "Einheit"  theory  that  all  streptococci 
were  identical,  used  a  single  strain  of  organism,  whose  virulence  was 
greatly  increased  for  rabbits  prior  to  injection  into  horses.  Immuniza- 
tion requires  several  months.  He  found  that  for  some  days  following 
each  injection  the  horse  exhibited  a  febrile  reaction,  and  during  that 
period  the  serum  was  toxic  for  rabbits;  streptococci  may  be  found  in 
the  blood  stream  during  this  period.  After  the  temperature  has 
reached  normal — three  weeks  or  more  after  the  injection — the  toxic 
properties  disappear  and  the  serum  exhibits  protective  powers  when 
it  is  introduced  into  rabbits  with  a  lethal  dose  of  streptococci.  This 
serum  has  been  used  extensively  in  the  treatment  of  erysipelas,  puer- 
peral fever,  and  scarlet  fever,  but  its  curative  value  is  still  a  matter 
of  discussion. 

Tavel's  serum  is  essentially  like  that  of  Marmorek,  except  that  a 
polyvalent  vaccine  is  used  for  immunization.  Besredka  also  uses  a 
polyvalent  vaccine  for  immunizing  horses,  but  the  organisms  are 
not  exalted  in  virulence  for  rabbits  by  passage  through  a  series  of 
them  before  inoculating  horses.  Besredka  believes  that  passage 
through  rabbits  may  modify  the  virulence  of  the  streptococci  for 
man,  from  whom  the  organisms  are  obtained  for  immunizing  the 
horses,  and  for  whom  the  serum  is  to  be  used.  Streptococcal  sera  are 
as  yet  of  debatable  value;  in  localized  lesions  they  have  frequently 
exhibited  some  therapeutic  value;  in  the  severe  generalized  infections 
in  man  they  are  usually  either  irregular  in  their  action  or  inactive. 
Somewhat  more  encouraging  results  have  been  reported  where  the 
specific  immune  serum  is  used  in  connection  with  autogenous  vaccines 
of  streptococci. 

Antibodies. — Agglutinins  are  present  in  the  sera  of  animals  immu- 
nized with  streptococcus  vaccines,  and  the  degree  of  agglutinating 
power  may  be  very  considerable  for  homologous  strains.  The  results 
are  usually  less  definite  with  heterologous  strains,  and  agglutinins 
developed  during  immunization  with  streptococci  are  of  no  consider- 
able value  in  prognosis.  The  part  they  may  play  in  immunization 
is  problematical. 

Complement  fixation  has  not  been  found  a  satisfactory  method  for 

1  Ann.  Inst.  Past.,  1895,  ix,  593. 

2  Loc.  cit. 


THE  STREPTOCOCCUS  GROUP  279 

identifying  streptococci;  the  results  are  occasionally  variable  without 
apparent  cause. 

Bacteriological  Diagnosis. — 1.  Microscopical  Examination. — Smears 
from  abscesses  or  inflammatory  areas  usually  exhibit  pairs  and  short 
chains  of  cocci  which  retain  the  Gram  stain.  Occasionally  the  organ- 
isms can  not  be  distinguished  with  certainty  from  staphylococci. 
Frequently,  when  microscopic  examination  fails  (and  this  is  usually 
the  case  when  blood  is  examined),  streptococci  are  found  by  cultural 
methods. 

2.  Cultural  Examination. — If  the  material  is  purulent,  it  may  be 
streaked  or  plated  out  on  0.1  per  cent,  dextrose  agar;  the  colonies  are 
small  and  transparent,  and  may  be  easily  overlooked.    Blood,  lymph 
or  serum  should  be  plated  on  blood  agar.    If  the  material  is  blood,  one 
part  may  be  added  to  two  parts  of  melted  plain  agar,  and  the  whole, 
after  thorough  mixing,  may  be  poured  into  sterile  Petri  dishes.    Usually 
small,  gray  colonies  with  relatively  broad,  clear  areas  of  hemolysis 
appear  within  forty-eight  hours.     If  lymph  and  serum  be. the  sus- 
pected material,  blood  agar  should  be  used  for  plating  out.    Hemolytic 
colonies,  as  above,  appear  usually  within  two  days.     It  is  always 
well  to  inoculate  1  or  2  c.c.  of  blood  serum  or  lymph  into  broth  and 
maintain  it  at  37°  C.  for  twenty-four  hours  to  enrich  the  culture,  then 
plate  on  blood  agar;  also  inoculate  a  like  amount  into  a  rabbit. 

3.  Animal  Inoculation. — The  intraperitoneal  injection  of  suspected 
fluids  into  rabbits  frequently  results  in  a  fatal  perftonitis,  from  which 
the  organism  may  be  recovered  from  the  blood  stream.     Relatively 
large  amounts  should  be  used. 

The  detection  of  streptococci  in  the  blood  of  a  patient  is  frequently 
an  unfavorable  clinical  sign;  it  does  not  necessarily,  however,  justify 
a  grave  prognosis.  Cases  are  met  with  which  present  symptoms  of 
septicemia,  yet  the  organisms  may  not  be  obtained  from  the  blood. 
Occasionally  the  patient  dies  from  toxemia,  due  apparently  to  the 
absorption  of  toxic  substances  from  the  local  infection.  Streptococci 
from  erysipelas,  septicemia,  scarlet  fever,  and  even  from  articular 
rheumatism  are  so  similar  culturally  and  morphologically  that  the 
various  strains  can  not  be  differentiated  with  certainty;  slight  varia- 
tions in  cultural  reactions  are  exhibited  by  all  these  strains.  Neither 
does  animal  experimentation  afford  definite  criteria  for  the  estab- 
lishment of  types.  Even  one  passage  through  an  animal  may  modify 
the  pathogenicity  greatly. 

In  the  light  of  our  present  knowledge  the  resistance  of  different 


280  STREPTOCOCCUS-PNEUMOCOCCUS  GROUP 

tissues  and  the  portal  of  entry  play  a  prominent  part  in  determining 
both  the  type  of  lesion  which  will  result  from  invasion  of  the  body 
by  streptococci,  and  the  modification  in  virulence  they  may  undergo 
in  man  or  animal  as  the  struggle  between  host  and  invader  is  extended 
in  time. 

Prophylaxis. — General  surgical  aseptic  methods.  Autogenous  vac- 
cines have  been  extensively  used  in  streptococcus  infections,  but  with 
less  favorable  results  than  autogenous  staphylococcus  vaccines. 

The  Streptococcus  Einheit  or  Vielheit. — Considerable  discussion 
has  arisen  concerning  the  unity  or  the  plurality  of  types  included 
within  the  organism  known  as  Streptococcus  pyogenes.  Marmorek1 
and  others  have  stoutly  maintained  the  Einheit  theory.  Considerable 


FIG.  36. — Pneumococcus  mucosus  showing  capsule.      X  1000. 

evidence  in  favor  of  this  view  was  advanced  by  Koch  and  Petruschky,2 
who  showed  that  a  streptococcus  obtained  from  a  fatal  puerperal 
sepsis  caused  erysipelas  in  a  rabbit  when  it  was  injected  subcutaneously, 
peritonitis  when  injected  intraperitoneally,  and  septicemia  when 
introduced  intravenously.  The  organisms  freshly  isolated  caused  a 
rapidly  fatal  septicemia  when  introduced  through  the  blood  stream, 
but  the  virulence  was  gradually  lost  following  cultivation  on  artificial 
media;  the  septicemic  phenomena  diminished  in  intensity  and  there 
was  evidence  of  a  localization  of  the  organisms.  Their  conclusions 
were  that  the  type  of  lesion  produced  by  Streptococcus  pyogenes 
depended  largely  upon  the  virulence  of  the  culture,  the  tissue  invaded, 
and  the  number  of  organisms.  With  a  comparatively  slight  loss  in 
virulence  the  endocardium  appeared  to  be  somewhat  more  frequently 

1  Berl.  klin.  Wchnschr.,  1902,  xxxix,  299. 

2  Loc.  cit. 


THE  STREPTOCOCCUS  GROUP  281 

the  site  of  the  focal  infection;  with  a  greater  loss  of  virulence,  the 
joints.  It  must  be  remembered  in  this  connection  that  the  virulence 
of  a  streptococcus  for  man  does  not  necessarily  determine  the  virulence 
for  animals. 

It  is  possible  to  raise  the  virulence  of  streptococci  very  materially 
by  artificially  creating  portals  of  entry  and  of  escape  which  are  not 
usually  available  to  the  streptococcus.  This  is  accomplished  by 
passage  through  experimental  animals.  By  passage  it  is  possible 
to  reproduce  with  considerable  accuracy  the  various  reactions  men- 
tioned above,  depending  upon  the  virulence  of  the  organism,  the 
tissue  into  which  the  injection  is  made,  and  the  number  of  organisms 
introduced.  It  is  also  important  to  remember  than  an  increase  in 
virulence  for  one  animal,  attained  by  frequent  passages,  frequently 
results  in  a  loss,  partial  or  complete,  of  the  virulence  of  the  organism 
for  another  animal.  Too  little  is  known  of  the  mechanism  of  virulence, 
however,  to  place  a  final  interpretation  upon  the  biological  signifi- 
cance of  changes  in  pathogenic  powers. 

Additional  evidence  of  the  Einheit  of  streptococci  has  been  brought 
forward  by  Rosenow,1  who  states  that  he  has  changed  streptococci 
to  pneumococci  and  back  again  by  special  methods  of  culture  and 
animal  inoculation.  Two  possibilities  present  themselves  to  explain 
this  phenomenon,  if  Rosenow's  claims  are  substantiated.  First,  the 
streptococcus-pneumococcus  complex  is  a  single  organism  which 
exhibits  nodes  of  relative  cultural  stability  (assuming  that  present- 
day  methods  for  the  recognition  of  bacterial  types  are  fundamentally 
sound),  and  the  organism  may  pass  from  one  node  to  another  under 
the  stress  of  environmental  stimuli.  The  second  possibility  is  that 
the  streptococcus  and  pneumococcus  are  in  reality  distinct  biological 
entities  and  that  an  actual  discontinuous  mutation  has  occurred. 
The  many  variables  to  be  considered  in  this  connection — variations 
in  virulence,  adaptability  to  various  hosts,  and  changes  in  appearance 
in  different  media,  all  of  which  may  change  independently  of  or  parallel 
to  each  other — complicate  the  problem  to  a  considerable  degree; 
final  judgment  must  await  the  establishment  of  authoritative  standards 
for  bacterial  diagnosis  of  unquestioned  fundamental  stability. 

Neufeld,  and  Cole  and  his  associates  have  presented  a  new  aspect 
of  the  problem.  They  found  that  the  older  conception  of  the  unity 
of  the  pneumococcus  type  was  untenable.  They  found  there  were 
four  distinct  types  of  pneumococcus  which  were  recognizable  both 

1  Loc.  cit. 


282  STREPTOCOCCUS-PNEUMOCOCCUS  CROUP 

by  serological  and  pathological  methods,  and  that  these  types  were 
mutually  stable,  for  long-continued  passage  through  animals  failed 
to  alter  or  modify  their  general  cultural  and  agglutinating  properties, 
although  the  virulence  of  the  respective  types  for  one  or  another 
animal  could  be  increased  or  decreased.  It  is  not  improbable  that  a 
thorough  study  of  the  streptococcus  group  may  reveal  similar  sero- 
logical variance  and  that  in  the  type  now  designated  Streptococcus 
pyogenes  several  individual  types  parallel  to  those  of  the  pneumococcus 
may  be  demonstrated. 

The  important  question  for  the  moment  is,  do  these  changes  of 
virulence,  et  cetera,  exhibited  by  the  streptococcus  influence  the  diag- 
nostic aspect  of  the  question?  Theobald  Smith  has  admirably  summed 
up  the  present  status  of  the  subject  in  the  following  words :  "  Spon- 
taneous changes  in  the  cultural  characters  of  the  streptococcus  do  not 
proceed  rapidly  enough,  if  they  go  on  at  all,  to  interfere  with  current 
bacteriological  methods.  Tendencies  toward  slow  changes  may  be 
used  as  further  valuable  distinguishing  characters."1 

THE   PNEUMOCOCCUS. 

Synonyms. — Micrococcus  pasteuri,  Diplococcus  pneumonia,  Diplo- 
coccus  lanceolatus,  Streptococcus  lanceolatus. 

Historical. — Although  the  pneumococcus  was  observed  by  Stern- 
berg2  and  independently  by  Pasteur3  in  the  blood  of  rabbits  inoculated 
with  sputum,  the  etiological  relationship  of  the  organism  to  lobar 
pneumonia  was  not  established  until  1886,  when  Frankel4  and  Weich- 
selbaum5  published  their  respective  studies  upon  lobar  pneumonia. 

Morphology. — Viewed  under  the  microscope,  the  pneumococcus 
presents  two  distinct  appearances,  depending  upon  the  source  of  the 
culture.  Observed  in  human  or  animal  tissues,  exudates  or  body 
fluids,  or  in  media  containing  non-coagulated  albuminous  fluids,  as 
blood  serum,  ascitic  or  hydrocele  fluids,  the  organisms  occur  typically 
in  pairs  surrounded  by  a  definite  capsule,  or  less  commonly  in  short 
chains  enclosed  in  a  capsule.  The  individual  cells  are  typically  lanceo- 
late in  shape  with  the  apposed  surfaces  of  each  pair  flattened,  and  the 
distal  ends  somewhat  pointed.  Less  commonly  the  organisms  are 
oval,  or  nearly  spherical.  The  paired  arrangement  is  maintained 
when  the  organisms  remain  adherent  to  form  short  chains.  Cultures 

1  Smith  and  Brown,  Jour.  Med.  Research,  1914,  xxxi,  501. 

2  National  Bureau  of  Health,  1881. 

3  Compt.  rend.  Acad.  Sci.,  1881,  xcii,  159. 

4  Zeit.  f.  klin.  Med.,  1886,  x,  401.     Ibid,  xi,  437. 
6  Wien.  med.  Jahrb.,  1886,  p.  483. 


THE  PNEUMOCOCCUS 

in  artificial  media  which  do  not  contain  albuminous  fluids  are  not 
encapsulated,  and  the  distinctive  lanceolate  shape  is  frequently  lost; 
the  organisms  become  more  nearly  oval  or  spherical  in  outline,  but 
the  tendency  to  remain  adherent  in  pairs  is  usually  maintained.  Chains 
of  from  four  to  eight  elements  are  developed  in  broth  cultures,  which 
has  led  many  observers  to  include  the  pneumococcus  in  the  strepto- 
coccus group.  The  size  of  the  organisms  varies  considerably;  ordi- 
narily the  lesser  diameter  measures  from  0.5  to  0.8  microns,  and  the 
longer  diameter  from  1  to  1.3  microns. 

The  pneumococcus  is  non-motile  and  possesses  no  flagella.  The 
capsule,  which  surrounds  pairs  of  organisms  derived  from  sputum, 
tissue,  body  fluids  and  exudates  of  man  and  animals,  as  well  as  those 


FIG.  37. — Pneumococcus  showing  capsules. 

cultivated  in  milk  or  media  containing  uncoagulated  albuminous  sub- 
stances, is  readily  demonstrated  by  the  methods  of  Welch,1  Hiss2  and 
Rosenow.3  The  capsule  is  poorly  formed  or  absent  from  pneumo- 
cocci  derived  from  chronic  processes  or  from  mucous  surfaces  where 
the  organisms  are  growing  as  parasites  or  "opportunists." 

The  ordinary  anilin  dyes  stain  pneumococci  readily,  and  they  are 
Gram-positive  when  freshly  isolated,  but  tend  to  become  Gram- 
negative  during  cultivation  in  artificial  media. 

Isolation  and  Culture. — Pneumococci  grow  slowly  and  feebly  upon 
ordinary  laboratory  media,  and  they  soon  perish.  Cultures  may 
be  obtained  from  the  blood  stream  in  a  large  percentage  of  cases  from 
the  fifth  day  of  the  disease  to  the  crisis4  by  inoculating  5  to  10  c.c.  of 

1  Bull.  Johns  Hopkins  Hospital,  1892,  xiii,  128. 

2  Cent.  f.  Bakt.,  Ref.,  1902,  xxxi,  302. 

3  Jour.  Infec.  Dis.,  1911,  ix,  1. 

4  Rosenow,  Jour.  Inf.  Dis.,  1904,  i,  280, 


284  STREPTOCOCCUS-PNEUMOCOCCUS  GROUP 

blood  into  100  to  150  c.c.  of  0.1  per  cent,  dextrose  broth,  and  incubating 
for  twenty-four  hours  at  37°  C.  Isolation  of  pneumococci  from  sputum 
by  cultural  methods  is  practically  hopeless;  but  pure  cultures  may 
be  obtained  from  the  heart  blood  of  white  mice  inoculated  subcutan- 
eously  with  sputum. 

The  organisms  may  be  obtained  from  inflammatory  exudates  and 
pus  either  by  inoculation  of  the  material  into  white  mice  or  infecting 
the  surface  of  blood  agar,  serum,  ascitic  or  hydrocele  agar  plates. 
Colonies  on  blood  agar  plates  are  minute,  gray,  and  surrounded  by  a 
greenish  halo  which  Butterfield  and  Peabody1  and  Cole2  have  shown 
to  be  methemoglobin.  Colonies  on  ascitic  agar  are  small,  transparent 
and  colorless.  The  growth  upon  plain  nutrient  agar  or  gelatin  is  very 
scanty.  Gelatin  is  not  liquefied.  The  addition  of  dextrose  to  agar 
increases  the  nutritive  value  of  the  medium,  but  the  acid  formed  by 
the  fermentation  of  the  dextrose  soon  kills  the  bacteria  unless  calcium 
carbonate  is  added  to  neutralize  the  acid.  Many  strains  of  pneumo- 
cocci grow  in  milk,  producing  as  a  rule  sufficient  acid  to  cause  coagula- 
tion. The  coagulum  is  never  liquefied.  Growth  upon  Loffler's  blood 
serum  is  moderately  luxuriant,  particularly  for  subcultures;  initial 
development  of  the  organisms  directly  from  human  or  animal  sources 
is  not  extensive  upon  this  medium.  The  colonies  are  small,  clear  and 
colorless,  and  not  distinctive.  Growth  is  more  rapid  in  fluid  than 
in  solid  media.  Secondary  inoculations  into  plain  broth  or  broth 
containing  utilizable  carbohydrates  result  in  a  clouding  of  the  medium 
and  extensive  development,  more  luxuriant  in  the  latter  than  the 
former.  The  addition  of  blood,  blood  serum  or  ascitic  fluid  to  media 
increases  the  nutritive  value  greatly.  The  organisms  die  within  a 
few  days,  and  even  after  twenty-four  hours'  incubation  degenerative 
forms  appear,  and  they  become  Gram-negative.  Transfer  at  frequent 
intervals  to  fresh  media  is  essential  to  maintain  viable  cultures  of  the 
pneumococcus. 

The  pneumococcus  is  an  aerobic,  facultatively  anaerobic  organism 
whose  limits  of  growth  lie  between  25°  C.,  below  which  development 
ceases,  and  about  42°  to  43°  C.;  the  optimum  temperature  of  growth 
is  37°  C.  The  organisms  are  not  resistant  to  heat,  being  killed  by  an 
exposure  of  ten  to  fifteen  minutes  to  55°  C.3  Chemical  disinfectants, 
as  5  per  cent,  carbolic  acid  or  1  to  1000  bichloride  of  mercury,  destroy 
pneumococci  readily.  Dried  rapidly  in  sputum,  they  retain  their 

1  Jour.  Exp.  Med.,  1913,  xvii,  587. 

2  Ibid.,  1914,  xx,  363. 

3  See  Wood,  Jour.  Exp.  Mod.,  1905,  vii,  592,  for  literature. 


THE  PNEUMOCOCCUS 


285 


viability  for  nearly  two  weeks,  but  sunlight  is  rapidly  fatal.  The 
virulence  is  rapidly  lost  during  cultivation  in  artificial  media,  but  it 
may  be  retained  practically  unimpaired  for  weeks  if  the  organisms 
suspended  in  blood  are  sealed  in  glass  tubes  and  maintained  in  the 
dark  at  ice-box  temperature.  Pneumococci  obtained  from  sputum, 
either  of  healthy  individuals  or  from  the  "rusty  sputum"  character- 
istic of  the  earlier  stages  of  lobar  pneumonia,  possess  sufficient  viru- 


FIG.  38. — Pneumococcus  in  sputum.      X  1000. 

lence  to  kill  white  mice.  The  original  virulence  may  frequently  be 
restored  to  cultures  on  artificial  media  by  passage  through  white 
mice,  provided  large  doses  are  administered  at  the  start.  Repeated, 
rapid  inoculations  of  virulent  pneumococci  frequently  lead  to  a  decided 
increase  of  virulence  above  that  originally  exhibited  by  the  organisms. 
Products  of  Growth. — Chemical. — The  pneumococcus  produces  acids, 
chiefly  lactic,  but  smaller  amounts  of  formic  acid,  in  hexoses,  bioses, 
and  many  starches.  Hiss1  has  shown  that  the  fermentation  of  inulin 
by  the  pneumococcus  is  a  very  constant  cultural  differentiation  of 
the  organism  from  the  streptococcus,  which  is  unable  to  ferment  this 
starch.  Another  important  method  of  distinguishing  between  pneumo- 
cocci and  streptococci  is  the  solubility  of  the  former  in  bile  or  a  freshly 
prepared  solution  of  sodium  chlorate.2  3  Colonies  of  the  pneumococcus 
on  blood  agar  are  surrounded  by  a  greenish  zone  of  methemoglobin.4 

1  Jour.  Exp.  Med.,  August,  1905,  vii.,  547. 

2  Neufeld,  Zeit.  f.  Hyg.,  1900,  xxxiv,  454.      Wadsworth,  Jour.  Med.  Research,  1904, 
x,  228. 

3  The  test  is  made  as  follows:     1  c.c.  of  a  twenty-four-hour  broth  culture  of  the  sus- 
pected organism  is  mixed  with  0.1  c.c.  of  a  freshly-prepared  2  per  cent,  solution  of  sod- 
ium chlorate  and  maintained  at  37°  C.      Clearing  of  the  solution  indicating  solution  of 
the  organisms  does  not  take  place  uniformly;  some  cultures  dissolve  more  rapidly  than 
others.    Cole,  Jour.  Exp.  Med.,  1912,  xvi,  658.    Acids  interfere  with  the  success  of  the 
test. 

4  Butterfield  and  Peabody,  loc.  cit.     Cole,  loc.  cit. 


286  STREPTOCOCCUS-PNEUMOCOCCUS  GROUP 

Enzymes  have  not  been  demonstrated  in  cultures  of  the  pneumo- 
coccus. 

Toxins. — Soluble  toxins  have  not  been  detected  in  cultures  of 
pneumococci,  although  the  filtrates  obtained  by  Klemperer,1  Wash- 
bourn,2  and  Isaeff3  were  toxic  for  small  laboratory  animals.  The 
toxicity  observed  in  these  preparations  was  probably  due  to  the 
liberation  of  endotoxins  as  the  result  of  autolysis  of  pneumococci  in 
the  medium.  Macfadyen4  has  obtained  toxic  substances  from  two- 
to  three-day  agar  cultures  of  virulent  pneumococci,  which  were 
ground  finely  after  freezing  with  liquid  air  (method  of  Macfadyen 
and  Roland),  then  extracted  with  1  to  1000  potassium  hydrate, 
centrifugalized  to  remove  fragments  of  the  organisms  and  filtered. 
A  small  amount  of  the  filtrate,  0.5  to  1  c.c.  in  rabbits,  0.1  to  1  c.c.  in 
guinea-pigs,  produced  death  when  injected  intravenously  or  intra- 
peritoneally.  The  toxicity  of  the  filtrate  was  roughly  proportional 
to  the  virulence  of  the  organisms  for  rabbits.  Heating  the  filtrate 
to  55°  C.  for  an  hour,  or  exposure  to  chloroform  vapor  for  the  same 
time  reduced  the  toxicity  of  the  preparation  very  considerably.  Neu- 
feld  and  Dold5  and  Rosenow6  obtained  toxic  substances  from  pneumo- 
cocci, the  former  by  extraction  of  the  organisms  in  0.1  per  cent, 
lecithin  in  physiological  salt  solution,  the  latter  by  simple  autolysis, 
which  induced  symptoms  in  guinea-pigs  suggesting  acute  anaphy- 
laxis.  Cole7  has  repeated  these  experiments  with  results  that  were 
irregular:  thus,  of  213  guinea-pigs  injected  with  extracts  of  pneumo- 
cocci in  salt  solution,  8  died  acutely  with  symptoms  resembling  acute 
anaphylactic  shock,  83  died  within  twelve  hours,  the  remainder  were 
negative.  Cole  concludes  that  extracts  of  pneumococci  in  salt  solu- 
tion may  be  toxic,  but  not  uniformly  so.  The  exact  conditions  under 
which  these  solutions  become  toxic  are  unknown.  Solutions  of 
pneumococci  dissolved  in  dilute  solutions  of  bile  salts  were  found  to 
be  very  constantly  toxic.8  The  intravenous  injection  of  these  solu- 
tions into  rabbits  and  guinea-pigs  elicits  symptoms  resembling  closely 
those  of  acute  anaphylaxis.  Many  of  the  animals  die  acutely. 


1  Zeit.  klin.  Med.,  1891,  xx,  165. 

2  Jour.  Path,  and  Bact.,  1897,  iii,  214. 
.  3  Ann.  Inst.  Past.,  1892,  vii,  259. 

4  Cent.  f.  Bakt.,  Orig.,  1907,  xliii,  30. 

5  Berl.  klin.  Wchnschr.,  1911,  xlviii,  1069. 

6  Jour.  Infec.  Dis.,  1911,  ix,  190. 

7  Jour.  Exp.  Med.,  1912,  xvi,  644. 

8  Casagrandi  (quoted  by  Pribram:   Kolle  and  Wassermann  Handb.,  2  ed.,  1913,  iia, 
1350)  states  that  normal  rabbit  blood  contains  antihemolysins. 


THE  PNEUMOCOCCUS  287 

Hemotoxin. — Recently  Cole1  has  shown  that  solutions  obtained  by 
dissolving  pneumococci  in  dilute  solutions  of  bile  salts,  or  by  tritura- 
tion,  are  hemolytic  for  rabbits,  guinea-pigs,  sheep  and  human  red  blood 
cells,  and  that  their  activity  is  inhibited  by  minute  amounts  of  choles- 
terin.  The  injection  of  these  solutions  in  gradually  increasing  amount 
leads  to  an  inhibition  of  their  action;  in  other  words,  this  "hemolytic 
endotoxin"  appears  to  act  as  an  antigen. 

Pathogenesis. —  Human. — At  least  90  per  cent,  of  all  cases  of  lobar 
pneumonia,  one  of  the  most  prevalent  and  fatal  of  human  diseases, 
is  caused  by  the  pneumococcus,  but  this  disease  is  by  no  means  the 
only  one  in  which  the  organism  is  an  etiological  factor.  Many 
bronchopneumonias  which  follow  acute  infections,  as  typhoid,  diph- 
theria, so-called  "  aspiration  pneumonia,"  are  also  of  pneumococcic 
causation.  Pleurisy,  a  frequent  complication  of  both  types  of  pneu- 
monia, is  quite  commonly  a  pneumococcus  infection,  and  a  majority 
of  sporadic  cases  of  meningitis,  particularly  in  children,  are  also  caused 
by  the  organism.  Indeed,  in  children  the  pneumococcus  is  rather 
more  commonly  isolated  from  suppurative  processes  than  any  other 
organism;  in  adults  the  incidence  of  pneumococci  in  suppurations  is 
on  the  whole  considerably  less.  Middle  ear  involvement,  inflamed 
mastoids,  endo-  and  pericarditis  are  all  frequently  caused  by  the 
pneumococcus.  The  channel  of  infection  appears  to  be  through  the 
blood  stream,  and  pneumococci  have  been  isolated  from  the  blood 
stream  in  a  very  large  percentage  of  all  cases  of  lobar  pneumonia.2 
Less  commonly  the  organisms  become  localized  in  joints,  causing 
arthritis,  and  around  the  shafts  of  bones,  causing  osteomyelitis. 
Conjunctival  inflammation  of  varying  degrees  of  severity  which 
occasionally  leads  to  ulcer  formation  is  frequently  a  pneumococcus 
infection. 

It  was  formerly  stated  that  virulent  pneumococci  could  be  obtained 
from  the  sputum  of  fully  30  per  cent,  of  normal  individuals.  The 
supposition  was  that  the  patient  became  the  victim  of  his  own 
organisms.  Recent  studies  by  Dochez  and  Avery3  suggest  strongly 
that  the  pneumococci  found  in  the  sputum  during  pneumonia  are 
commonly  replaced  by  pneumococci  of  a  less  virulent  type  soon  after 
convalescence.  Their  observations,  furthermore,  make  it  justifiable 
to  consider  those  patients  who  harbor  the  more  virulent  types  after 

1  Jour.  Exp.  Med.,  1914,  xx,  346. 

2  Rosenow,  loc.  cit. 

3  Quoted  by  Cole,  New  York  Med.  Jour.,  January  2  and  9,  1915. 


288  STREPTOCOCCUS-PNEUMOCOCCUS  GROUP 

recovery  as  carriers,  precisely  as  typhoid  carriers  harbor  typhoid 
bacilli  after  recovery  from  typhoid  fever. 

Animal. — Mice  are  the  most  susceptible  of  laboratory  animals 
to  infection  with  the  pneumococcus.  Small  amounts  of  pneumonic 
sputum,  exudate  or  pus  injected  subcutaneously  lead  to  a  rapidly  fatal 
septicemia.  Encapsulated  pneumococci  are  found  in  the  blood  and 
visceral  organs,  particularly  the  spleen,  which  is  enlarged,  and  the 
peritoneal  fluid.  Rabbits  are  somewhat  less  susceptible  and  the 
results  of  inoculation  of  pneumococcic  exudates  or  cultures  depend 
upon  the  virulence  of  the  organisms,  the  size  of  the  dose,  and  the 
method  of  inoculation.1  The  intravenous  or  subcutaneous  inoculation 
of  virulent  cultures  leads  to  a  fatal  septicemia,  death  occurring  within 
five  days  as  a  rule.  The  less  virulent  organisms,  which  do  not  kill 
the  animal  within  a  few  days  after  inoculation,  frequently  cause 
localized  abscess  formation  with  a  fibrinous  exudate.  The  nature 
and  extent  of  the  lesions  induced  depend  largely  upon  the  time  which 
elapses  between  inoculation  and  the  death  of  the  animal.  In  general 
it  may  be  stated  that  localized  lesions  appear  when  less  virulent 
organisms  are  injected.  Intravenous  injections  are  more  effective 
than  subcutaneous  inoculations  of  the  same  amount  of  organisms. 
Guinea-pigs  are  relatively  non-susceptible  to  pneumococcus  infection. 

Many  attempts  have  been  made  to  reproduce  the  typical  patho- 
logical lesions  of  lobar  pneumonia  in  experimental  animals.  Wads- 
worth2  succeeded  in  reproducing  typical  lobar  pneumonia  in  rabbits 
by  first  partially  immunizing  them  to  the  organism  in  order  to  localize 
the  lesions  in  the  lungs.  Lamar  and  Meltzer,3  and  Wollstein  and 
Meltzer4  produced  lobar  pneumonia  in  dogs  by  the  method  of  tracheal 
insufflation  devised  by  Meltzer;  and  Winternitz  and  Hirschf elder5 
have  been  equally  successful  in  producing  lobar  pneumonia  in  rabbits. 
The  method  consists  essentially  in  forcing  suspensions  of  pneumo- 
cocci deep  into  the  terminal  bronchioles  and  their  alveoli.  Cole6 
has  shown  that  the  strain  of  organism  influences  the  results ;  organisms 
of  slight  virulence  give  negative  results,  and  organisms  possessing  too 
great  virulence  cause  a  generalized  septicemia  with  congestion  and 
edema  of  the  lungs  as  the  only  local  pulmonary  manifestations. 

1  Kruse  and  Pansini,  Zeit.  f.  Hyg.,  1892,  xi,  279  et  scq. 

2  Am.  Jour.  Med.  Sc.,  1904,  cxxvii,  851. 

3  Jour.  Exp.  Med.,  1912,  xv,  133. 

4  Ibid.,  1913,  xvii,,353,  424. 

5  Ibid.,  1912,  xvii,  657. 

6  Arch.  Int.  Med.,  1914,  xiv,  56. 


THE  PNEUMOCOCCUS 


289 


Types  of  Pneumococci. — Kruse  and  Pansini1  as  early  as  1891  called 
attention  to  the  differences,  both  cultural,  morphological  and  in 
virulence,  which  they  observed  in  studying  eighty-four  strains  of 
pneumococci  isolated  from  many  cases  of  pneumonia.  They  believe 
that  there  was  no  sharp  line  of  demarcation  between  the  pneumo- 
coccus  and  Streptococcus  pyogenes,  because  their  various  strains 
included  all  variants  between  the  two  types  of  organisms.  Recently 
Rosenow2  has  reported  the  transmutation  of  typical  pneumococci  to 
Streptococcus  pyogenes  by  a  series  of  animal  passages  and  cultural 
manipulations.  Cole3  has  been  unable  to  confirm  this  observation 
in  any  one  of  several  hundred  strains,  but  it  should  be  stated  that 
he  has  not  employed  Rosenow's  procedure  in  detail. 

Much  light  has  been  shed  upon  the  apparent  variability  of  strains 
of  pneumococci  by  the  observations  of  Neufeld  and  Handel,4  and 
Dochez,3  and  Dochez  and  Gillespie.6  These  observers  have  shown 
by  serological  reactions  that  pneumococci  may  be  divided  into  four 
groups  or  types,  each  of  which  fails  to  agglutinate  with  sera  other 
than  the  homologous  serum.  These  groups  have  been  tentatively 
designated  I  to  IV  inclusive.  Groups  I  and  II  are  typical  virulent 
pneumococci.  Group  III  comprises  the  organism  formerly  known 
as  Streptococcus  mucosus,  now  called  Pneumococcus  mucosus;  and 
Group  IV  includes  relatively  avirulent  strains  which  are  commonly 
found  in  the  mouths  of  healthy  persons.  Group  IV  is  somewhat 
more  heterogenous,  judging  from  agglutination  reactions,  than  Groups 
I  to  III.  Group  III  contains  the  most  virulent  organisms.  A  study 
of  the  distribution  of  the  various  types  in  seventy-two  cases  of  pneu- 
monia illustrates  this  point.7 


Infection  type. 
1 

2     ..• 

3 

4 


Total 


No.  cases. 
34 
13 
10 
15 

72 


No.  deaths. 
8 
8 
6 

1 

23 


Per  cent. 
24 
61 
60 

7 

32 


It  is  possible  that  "  mixed  infections"  will  be  found  when  more 
cases  are  carefully  studied.  The  same  general  types  have  since  been 
reported  in  Europe  and  in  Philadelphia.8 

1  Loc.  cit.  2  Jour.  Am.  Med.  Assn.,  1913,  Ixi,  2007.  3  Loc.  cit. 

4  Zeit,  f.  Immunitatsforsch.,  1909,  iii,  159;  Berl.  klin.  Woch.,  1912,  xlix,  680. 

5  Jour.  Exp.  Med.,  1912,  xvi,  680.  6  Jour.  Am.  Med.  Assn.,  1913,  Ixi,  727. 
*  Cole,  Arch.  Int.  Med.,  1914,  xiv,  33. 

8  Cole,  New  York  Med.  Jour.,  January  2  and  9,  1915. 
19 


290  STREPTOCOCCUS-PNEUMOCOCCUS  GROUP 

Immunity  and  Immunization. — Relatively  little  is  known  of  the 
nature  and  extent  of  immunity  following  recovery  from  an  attack 
of  pneumonia.  One  attack  appears  to  predispose  somewhat  to  a  sub- 
sequent attack,  which  was  explained  formerly  on  the  basis  that  little 
or  no  immunity  was  conferred  on  the  patient.  The  extensive  work 
of  Cole  and  his  associates  suggests  that  a  second  attack  of  the  disease 
may  be  caused  by  a  different  type  of  pneumococcus;  their  experiments 
indicate  that  antibodies  specific  for  one  type  are  not  protective  against 
infection  with  the  other  types. 

The  serum  of  convalescent  pneumonia  patients  exhibits  relatively 
feeble  bactericidal  activity,  even  upon  the  homologous  strain  of  the 
pneumococcus,  and  the  mechanism  which  leads  to  recovery  is  not 
definitely  known.  Neufeld1  and  others  have  advanced  the  hypothesis, 
based  upon  careful  observation,  that  the  crisis  in  pneumonia,  which 
usually  marks  the  end  of  the  prominent  clinical  symptoms,  is  asso- 
ciated with  a  somewhat  abrupt  increase  in  the  amount  of  specific 
opsonin  of  the  blood — an  increase  in  bacteriotropins  in  Neufeld's 
terminology.  This  theory  assumes  that  leukocytes  play  a  prominent 
part  in  the  healing  process,  and  that  phagocytic  activity  becomes 
efficient  at  or  about  the  time  of  the  crisis. 

Neufeld  and  Handel,2  and  Cole  and  his  associates3  have  produced  a 
serum  which  protects  susceptible  animals,  as  mice,  against  many 
times  the  fatal  dose  of  the  homologous  strain  of  organism  by  injecting 
gradually  increasing  doses  of  very  virulent  pneumococci  into  horses. 
Cole  has  used  these  sera  clinically  in  the  treatment  of  pneumonia 
with  promising  results  in  infections  caused  by  Types  I  and  II  of  the 
pneumococcus.  The  serum  appears  to  destroy  or  greatly  reduce  the 
number  of  pneumococci  in  the  blood,  and  to  be  of  material  benefit  in 
reducing  the  severity  of  the  infection.  At  present  a  satisfactory 
serum  for  infection  with  Type  III,  Pneumococcus  mucosus,  has  not 
been  prepared.  Cole  specifically  directs  attention  to  the  necessity 
of  identifying  the  type  of  infecting  organism  (by  agglutination  reac- 
tions) before  administering  the  serum.  It  is  imperative  that  the 
homologous  serum  be  used. 

Bacteriological  Diagnosis. — Pneumococci  are  found  in*  the  healthy 
throats  of  a  very  considerable  percentage  of  adults,  consequently  the 
identification  of  pneumococci  in  the  sputum  is  of  little  clinical  signifi- 

1  Zeit.  f.  Immunitatsforsch.,  1909,  iii,  159. 

2  Arb.  a.  d.  kais.  Gesamte,  1910,  xxxiv,  169. 

3  Jour.  Am.  Med.  Assn.,   1913,  Ixi,  663;  New   York  Med.  Jour.,  January  2  and   9, 
1915. 


THE  PNEUMOCOCCUS  291 

cance  unless  the  type  of  the  organism  is  determined.  Dochez  and 
Avery1  have  found  that  the  common  mouth  pneumococcus  is  usually 
the  avirulent  type  (Type  IV);  convalescents  from  pneumonia  usually 
exhibit  the  virulent  types,  I  to  III,  as  a  rule.  These  types  can  be 
identified  by  agglutination  reactions  with  the  specific  sera  prepared  by 
Cole. 

Pneumococci  isolated  from  pleural  and  pericardial  exudates,  middle- 
ear  infection,  empyema  and  pneumococcic  cerebrospinal  meningitis 
can  be  identified  morphologically  by  their  lanceolate  shape  and  Gram- 
positiveness ;  the  type  of  organism,  however,  must  be  determined  by 
serological  reactions. 

They  are  best  obtained  in  pure  culture,  if  they  are  mixed  with  other 
bacteria,  upon  blood  agar  plates.  A  green  halo  surrounds  the  typical 
pneumococcus  colony. 

The  prophylaxis  is  the  same  as  for  any  acute  respiratory  disease. 

1  Quoted  by  Cole,  loc.  cit. 


CHAPTER  XIV. 
THE  MENJNGOCOCCUS— GONOCOCCUS  GROUP. 

THE  MENINGOCOCCUS  GROUP.  THE  GONOCOCCUS  GROUP. 

Micrococcus  Meningitidis.  Micrococcus  Gonorrhrae. 

Parameningococcus.  Micrococcus  Catarrhalis. 

THE  MENINGOCOCCUS   GROUP. 

Micrococcus  Meningitidis.— Synonyms. — Diplococcus  intracellularis 
meningitidis;  Diplococcus  weichselbaumii,  Meningococcus. 

Historical. — Micrococcus  meningitidis  was  isolated  in  pure  culture 
by  Weichselbaum1  from  purulent  cerebrospinal  fluids  of  several  typical 
cases  of  cerebrospinal  meningitis.  The  injection  of  pure  cultures  of 
the  organisms  directly  into  the  meninges  of  dogs  resulted  in  well- 
marked  meningeal  inflammation  and  encephalitis.  Other  organisms, 
pneumococci,  streptococci,  Bacillus  influenzse,  for  example,  may  incite 
inflammations  of  the  cerebrospinal  membranes,  but  these  bacteria 
do  not  ordinarily  cause  epidemics  of  the  disease.  The  meningococcus 
frequently  causes  wide-spread  infection,  and,  unlike  the  organisms 
just  mentioned  (except  the  pneumococcus  occasionally)  the  typical 
lesions  are  primarily  of  the  cerebrospinal  axis. 

Morphology. — Meningococci  obtained  directly  from  the  cerebrospinal 
fluid  or  from  meningeal  exudates  occur  characteristically  in  pairs  with 
their  apposed  sides  flattened  and  somewhat  elongated.  They  measure 
about  one  micron  in  diameter,  although  the  size  varies  even  in  the 
same  culture.  The  individuals  are  fairly  uniform  in  size  and  shape 
in  very  young,  fresh  cultures,  but  in  older  cultures  considerable 
variations  in  size  are  met  with.  Examined  directly  in  inflammatory 
exudates  from  the  spinal  fluid  or  meninges  during  the  acute  stages  of 
the  disease,  the  organisms  occur  typically  and  characteristically  as 
intra-  and  extracellular  diplococci  and  tetrads.  They  are  found  in 
polymorphonuclear  leukocytes,  but  never  in  lymphocytes  or  other 
body  cells.2  They  are  intracellular  but  never  intranuclear,  according 
to  Councilman,  Mallory  and  Wright. 

1  Fortschr.  d.  Med.,  1887,  Nos.  18  and  19. 

2  Councilman,  Mallory  and  Wright,  Epidemic  Cerebrospinal  Meningitis.      A  Report 
to  the  Mass.  St.  Bd.  of  Health,  1898,  p.  75. 


THE  MENINGOCOCCUS  GROUP  293 

The  organisms  are  non-motile  and  possess  no  flagella.  No  spores 
are  forjned  and  no  capsules  have  been  demonstrated.  (Jaeger1 
believed  that  the  organisms  produced  capsules,  but  his  observations 
are  unconfirmed.)  Ordinary  anilin  dyes  stain  meningococci,  but  quite 
irregularly.  Occasionally  one  element  of  a  pair  stains  intensely 
while  its  fellow  stains  faintly  or  .not  at  all.  Relatively  large  oval 
or  round  forms  are  frequently  seen  in  cultures  and  in  purulent  exudates 
as  well,  which  exhibit  a  brightly  staining  point  in  the  centre  of  the 
organism;  the  remainder  of  the  cell  is  scarcely  colored.2  Carbol- 
thionin  is  one  of  the  best  stains  for  the  organism.  The  meningococcus 


FIG.  39. — Meningococci  in  pus.      X  1000. 

is  Gram-negative.  Meningococci  obtained  from  purulent  exudates 
or  from  cultures  on  artificial  media  can  not  be  definitely  differentiated 
from  gonococci  or  even  from  Micrococcus  catarrhalis  by  any  known 
staining  methods.  The  source  of  the  material  should  be  known  before 
even  a  tentative  morphological  diagnosis  is  attempted. 

Isolation  and  Culture. — The  meningococcus  grows  feebly  or  not  at 
all  upon  ordinary  artificial  media.  Growths  may  be  obtained  upon 
agar  containing  animal  protein,  as  defibrinated  blood  or  ascitic  fluid, 
or  upon  Loffler's  blood  serum  by  smearing  cerebrospinal  fluid  (drawn 
with  aseptic  precautions  by  lumbar  puncture)  in  liberal  amounts 
upon  the  surface  of  these  media.3  The  addition  of  1  per  cent,  of 
dextrose  to  the  media  favors  development  of  the  cocci.  If  the  fluid 
obtained  is  not  turbid,  centrifugalization  should  be  resorted  to  and 

1  Zeit.  f.  Hyg.,  1895,  xix,  358. 

2  Councilman,  Mallory  and  Wright,  loc.  cit.,  p.  74. 

3  For  technic  of  lumbar  puncture,  see  page  226. 


294  THE  MENINGOCOCCUS—GONOCOCCUS  GROUP 

the  sediment  distributed  as  densely  as  possible  in  the  manner  indi- 
cated. A  few  small,  transparent,  round  colonies  are  usually  obtained 
when  relatively  large  amounts  of  material  are  inoculated.  The  first 
growth  upon  artificial  media  is  difficult  to  obtain;  secondary  trans- 
fers, if  made  within  three  days  from  initial  cultivations,  are  usually 
successful  and  development  is  somewhat  more  vigorous.  It  should 
be  emphasized  that  relatively  large  amounts  of  cocci  must  be  inocu- 
lated to  insure  growth  in  artificial  media.1  Little  or  no  growth  occurs 
in  plain  broth;  the  addition  of  calcium  carbonate2  to  dextrose  broth 
makes  a  favorable  medium  for  the  development  of  the  organism. 
Ascitic  and  serum  broths  are  suitable  media  for  the  meningococcus. 
A  coherent  sediment  gradually  accumulates  in  these  media  and  a 
delicate  pellicle  usually  forms  on  the  surface  after  a  few  days.  Secon- 
dary transfers  in  milk  usually  grow,  but  there  is  little  or  no  detectable 
change  in  the  physical  properties  of  the  medium. 

The  meningococcus  is  essentially  an  aerobic  organism,  at  least  in 
its  development  outside  the  human  body.  The  optimum  tempera- 
ture of  growth  is  37°  C.,  and  growth  ceases  when  the  temperature 
exceeds  42°  C.  or  falls  below  25°  C.  The  organism  is  soon  killed  by 
low  temperatures.  Stock  cultures  can  not  be  maintained  at  the 
temperature  of  the  ice-box;  they  should  be  kept  at  temperatures 
between  32°  and  38°  C.  Frequent  transfers  (every  two  or  three  days) 
must  be  made  to  maintain  the  viability  of  the  organism ;  exceptionally 
strains  are  met  with  which  become  acclimatized  to  the  conditions 
obtaining  in  artificial  media  to  such  a  degree  that  transfers  made  at 
less  frequent  intervals  suffice  to  maintain  the  viability  of  the  culture. 

The  meningococcus  exhibits  little  resistance  to  heat,  drying  or  the 
action  of  chemical  agents.  Five  minutes'  exposure  to  65°  C.  or  two 
minutes'  exposure  at  80°  C.  suffices  to  sterilize  the  culture.  Drying 
for  a  few  hours  at  20°  C.  is  likewise  fatal  to  the  organism.  Exposure 
of  the  organism  to  carbolic  acid  broth  (1  to  800)  inhibits  development, 
and  drying  in  the  dark  for  seventy- two  hours  is  fatal;  sixty  hours' 
exposure  to  drying  is  insufficient  to  kill  the  organisms.3 

Products  of  Growth. — Meningococci  are  culturally  very  inert.  No 
proteolytic  enzymes  have  been  demonstrated;  gelatin  and  blood 
serum  are  not  liquefied,  and  no  coagulation  or  peptonization  of  milk 
occurs.  Indol,  skatol,  phenol  or  other  products  of  similar  nature  are 

1  The  organisms,  like  gonococci,  degenerate  rapidly  in  artificial  media.     This  may 
explain  the  necessity  of  transferring  the  organisms  at  frequent  intervals. 
*  Bolduan,  New  York  Med.  Jour.,  1905,  May  13. 
3  Councilman,  Mallory  and  Wright,  loc.  cit.,  p.  78. 


THE  MENINGOCOCCUS  GROUP  295 

not  demonstrable  in  cultures  of  Jhe  organism.  Acid,  but  no  gas,  is 
produced  with  considerable  regularity  in  dextrose  and  maltose  broths;1 
other  ordinary  carbohydrates  are  unattacked.  These  fermentation 
reactions  are  of  considerable  value  in  the  cultural  differentiation  of 
meningococci  from  other  organisms  which  may  readily  be  confused 
with  them. 

Toxins. — Soluble  exotoxins  have  never  been  demonstrated  among 
the  products  produced  by  the  meningococcus;  killed  cultures  of  the 
organism  appear  to  be  as  fatal  for  ordinary  experimental  animals 
as  the  living  organisms.  This  would  suggest  that  the  toxic  phenomenon 
may  be  attributable  to  the  liberation  of  endotoxins  rather  than  to  a 
soluble  toxin. 


FIG.  40. — Meningococci  from^ferebrospinal  fluid.      X  1200.     (Kolle  and  Hetsch.) 

Pathogenesis. — The  meningococcus  possesses  but  feeble  pathogenic 
powers  for  guiiffea^pigs ;  all  attempts  to  induce  infection  by  subcuta- 
neous injections,  according  to  Councilman,  Mallory  and  Wright,2 
were  negative.  Occasionally  successful  results  were  obtained  from 
intraperitoneal  and  intrapleural  inoculation.  A  slight  fibrinopurulent 
exudate  was  found  postmortem  in  the  peritoneal  or  pleural  cavities 
in  the  fatal  cases.  Intracranial  inoculations  were  uniformly  negative. 
One  successful  infection,  of  a  goat  by  spinal  canal  inoculation  was 
obtained  by  these  observers;  the  animal  died  within  twenty-four 
hours,  and  autopsy  revealed  intense  congestion  of  the  meninges  of 
the  cord  and  brain.  A  small  amount  of  purulent  spinal  fluid  was 

1  Kopetsky,  Meningitis,  The  Laryngoscope,  1912,  xxii,  797,  has  called  attention  to  the 
early  disappearance  of  the  reducible  substance  (dextrose?)  normally  present  in   the 
spinal  fluid  in  cerebrospinal  meningitis.     It  is  possible  that  the  action  of  the  organism 
upon  this  substance  explains  the  phenomenon. 

2  Loc.  cit.,  p.  76. 


296  THE  MENINGOCOCCUS— GONOCOCCUS  GROUP 

obtained  containing  but  little  fibrin.  Small  numbers  of  cocci  were 
found  within  the  polymorphonuclear  leukocytes.  Flexner1  and  Von 
Lingelsheim  and  Leuchs2  have  reproduced  the  essential  lesions  of 
cerebrospinal  meningitis  in  monkeys  by  the  subdural  injection  of 
suspensions  of  the  organisms.  The  organisms  were  recovered  in  pure 
culture  at  autopsy. 

The  evidence  of  the  etiological  relation  of  the  meningococcus  to 
cerebrospinal  meningitis  in  man  is  essentially  the  common,  almost 
constant  demonstration  of  meningococci  in  the  cerebrospinal  fluid 
and  exudates  antemortem,  and  from  the  tissues  of  the  brain  and  cord 
postmortem.  It  must  be  remembered  that  other  organisms  can 
produce  essentially  the  same  lesions,  however.  The  nature  and  extent 
of  the  lesions  observed  in  fatal  cases  varies  somewhat  with  the  time 
which  elapses  between  the  onset  of  symptoms  and  death.  The  rapidly 
fatal  cases  frequently  exhibit  intense  congestion  of  the  membranes 
of  the  cord  and  brain;  usually  a  fibrinopurulent  exudate  forms, 
more  extensive  as  a  rule  at  the  base  of  the  brain  but  readily  demon- 
strable in  the  spinal  fluid  obtained  by  lumbar  puncture.  According 
to  Westenhoffer,3  there  is  commonly  a  swelling  of  the  tonsils  and 
pharynx  in  the  early  stages  of  the  disease;  middle  ear  involvement 
is  comparatively  frequent.  It  is  probable  that  the  organism  passes 
from  the  nose  and  nasopharynx  through  the  lymphatics  to  the  base 
of  the  brain.  The  accessory  sinuses  of  the  nasal  cavity  appear  to  be 
inflamed  in  a  majority  of  cases,  particularly  during  the  initial  clinical 
period  of  the  disease.  There  is  a  thickening  of  the  meninges  in  those 
cases  which  run  a  more  chronic  course,  frequently  with  considerable 
distention  of  the  ventricles.  Intracranial  pressure  is  usually  a  promi- 
nent symptom.  The  organism  has  been  isolated  from  the  blood  by 
Jacobitz,4  Dieudonne,5  Elser,6  Elser  and  Huntoon,7  the  latter  in  25 
per  cent,  of  their  large  series  of  cases. 

Immunity  and  Immunization. — Little  is  definitely  known  of  man's 
immunity  to  the  meningococcus.  One  of  the  surprising  results  of  the 
intensive  study  of  the  epidemic  disease  is  the  occurrence  of  the  organ- 
ism in  the  nasopharynx  in  a  very  considerable  number  of  apparently 
healthy  individuals,  chiefly  among  those  in  actual  contact  with 
patients,  less  commonly  among  those  not  intimately  in  association 
with  cases  but  in  regions  where  the  disease  is  epidemic,  and  rarely 

1  Cent.  f.  Bakt.,  1907,  xliii,  99.  2  Klin.  Jarhb.,  1906,  xv,  489. 

3  Berl.  med.  Gesellsch.,  1905,  May  17;  abstr.  Cent.  f.  Bakt.,  Ref.,  1905,  xxxvi,  754. 

4  Munchen.  med.  Wchnschr.,  1905.  *  Cent.  f.  Bakt.,  Orig.,  1906,  xli,  420. 
6  Jour.  Med.  Research,  1906,  xiv,  89.  7  Jour.  Med.  Research,  1909,  xx,  371. 


THE  MENINGOCOCCUS  GROUP  297 

among  individuals  residing  in  areas  where  but  few  sporadic  cases 
have  been  reported.  The  percentage  of  positive  examinations  varies 
considerably.  Dieudonne1  found  about  12  per  cent,  of  normal  soldiers 
in  a  garrison  at  Munich,  where  an  outbreak  occurred,  gave  positive 
cultures  from  the  nasopharynx.  Bruns  and  Hohn2  found  465  carriers 
among  3154  healthy  individuals  in  a  community  where  the  disease 
was  epidemic.  They  also  found  the  percentage  of  carriers  was  great- 
est when  the  epidemic  was  at  its  height.  Usually  these  carriers  are 
temporary  carriers;  smaller  numbers  become  permanent  carriers  or 
periodic  carriers.3 

Serum  Therapy.— Many  attempts  have  been  made  to  prepare  sera 
for  the  treatment  of  epidemic  cerebrospinal  meningitis,  and  two 
preparations  have  stood  the  test  of  actual  practice,  Kolle  and  Wasser- 
mann's4  serum  and  the  serum  prepared  by  Flexner  and  Jobling.  The 
method  of  immunization  adopted  by  Flexner  and  Jobling  appears 
from  available  data  to  be  essentially  that  of  Wassermann.  It  is  as 
follows:  horses  are  injected  subcutaneously,  first  with  dead  cultures 
of  meningococci,  secondly  with  live  cultures,  and  finally  with  auto- 
ly sates  of  cultures.  The  latter  are  prepared  by  suspending  virulent 
meningococci  in  sterile  water  for  two  days  at  37°  C.  and  injecting 
the  supernatant  fluid.  The  serum  thus  produced  appears  to  combine 
phagocytic  properties,  increasing  the  destruction  of  the  organisms  by 
leukocytes;  bacteriolytic  properties,  killing  and  dissolving  the  cocci, 
and  possibly  some  antitoxic  properties  as  well.  It  is  essential,  as 
Flexner  has  pointed  out,  to  inject  the  serum  directly  into  the  spinal 
canal.  This  is  accomplished  by  lumbar  puncture.  The  turbid  spinal 
fluid  is  allowed  to  escape  through  the  needle  with  which  the  puncture 
is  made  until  symptoms  of  intercranial  pressure  are  reduced.  An 
additional  amount  of  fluid  is  then  withdrawn  to  make  way  for  the 
serum  which  is  injected  directly,  15  to  20  c.c.  for  young  children  and 
20  to  40  c.c.  for  adults.  The  treatment  is  repeated  from  two  to  several 
times,  until  the  spinal  fluid  is  clear  and  has  a  normal  appearance  and 
cellular  content.  The  serum  must  be  used  early  in  the  disease  to 
obtain  the  best  results.  Flexner  and  Jobling6  have  analyzed  328 
cases  with  the  following  mortality : 

Per  cent. 

Injection  during  first  to  third  day  of  disease mortality  19.9 

Injection  during  fourth  to  seventh  day  of  disease     ....     mortality  22.0 
Injection  after  seventh  day  of  disease mortality  36.4 

1  Loc.  cit.  2  Klin.  Jahrb.,  1908,  xviii,  285. 

3  Mayer  and  Waldmann,  Munch,  med.  Wchnschr.,  1910,  475.  Mayer,  Waldmann, 
Furst  and  Gruber,  Munchen.  med.  Wchnschr.,  1910,  1584. 

<  Deut.  med.  Wchnschr.,  1906.  5  jour.  Am.  Med.  Assn.,  1908,  li,  No.  4. 


298  THE  MENINGOCOCCUS—GONOCOCCUS  GROUP 

Similar  results  have  been  obtained  in  Germany  with  Wassermann's 
serum.1  Later  observations  by  Flexner2  confirm  these  results.  The 
mortality  has  been  reduced  from  about  70  per  cent,  to  about  20  to 
25  per  cent. 

Bacteriological  Diagnosis. — (a)  Morphological. — The  demonstration  of 
Gram-negative,  biscuit-shaped  diplococci  in  purulent  spinal  fluid  from 
patients  exhibiting  the  characteristic  clinical  symptoms  is  sufficient 
to  establish  a  diagnosis  of  the  meningococcus.  It  is  to  be  remem- 
bered that  the  spinal  fluid  is  clear  for  the  first  twenty-four  hours  of 
the  disease,  and  usually  clear  after  the  tenth  day  to  the  fourteenth 
day  even  in  untreated  cases.  Centrifugalization  in  sterile  tubes  must 
be  resorted  to  in  such  cases;  the  sediment  is  examined  as  above. 
Smears  from  the  nasopharynx,  from  middle-ear  infections,  and  from 
suspected  carriers  can  not  be  definitely  diagnosed  upon  morphological 
characters  alone.  Cultural  characteristics  must  be  studied  as  well. 

Cultural  Characters. — Spinal  fluid  removed  aseptically  (and  cen- 
trifugalized  if  the  fluid  is  clear)  and  material  from  the  nasopharynx, 
nasal  cavity,  or  accessory  nasal  sinuses3  is  spread  upon  Loffler's 
blood  serum  and  incubated  at  37°  C.  After  twenty-four  to  forty-eight 
hours'  incubation,  small,  clear,  round  colonies  develop  in  the  majority 
of  cases  in  which  meningococci  are  present.  These  should  be  trans- 
ferred to  ascitic  broth  (preferably  containing  1  per  cent,  of  dextrose 
and  a  small  piece  of  calcium  carbonate)  and  examined  after  twenty- 
four  hours'  incubation  at  body  temperature.  If  growth  occurs,  inocu- 
lation should  be  made  in  ascitic  fluid  dextrose  and  ascitic  fluid  maltose 
broths  to  determine  if  acid  is  produced.  Several  diplococci  have  been 
found  which  resemble  the  meningococcus  microscopically  but  which 
differ  from  it  in  their  fermentation  reactions.  A  negative  result  does 
not  exclude  the  possibility  of  an  infection  with  the  meningococcus; 
negative  cultures  occur  quite  frequently.  Von  Lingelsheim4  and  Elser 
and  Huntoon5  have  studied  these  organisms  carefully  and  give  the 
following  differential  table : 


1  Wassermann,  Deut.  med.  Wchnschr.,  1907,   1585;  Wassermann  and  Leuchs,  Klin. 
Jahrb.,  1908,  xix,  Heft  3. 

2  Jour.  Am.  Med.  Assn.,  1909,  liii,  1443. 

3  Material  for  examination  from  the  nasopharynx  is  best  obtained  upon  sterile  swabs; 
the  infected  swab  should  be  immediately  rubbed  over  the  surface  of  a  series  of  blood 
serum  tubes  or  ascitic  agar  plates.  This  method  is  particularly  adapted  for  the  exami- 
nation of  suspected  carriers. 

4  Klin.  Jahrb.,  1906,  xv,  Heft  2. 

6  Jour.  Med.  Research,  1909,  xx,  377. 


THE  MENINGOCOCCUS  GROUP  299 


O  Q  3  O  S  ^  % 

Meningococcus —  +                                  + 

Pseudomeningococcus —  +                                  + 

Gonococcus —  + 

Micrococcus  catarrhalis —  — 

Diplococcus  crassus3 +  +  +          +          +          +          + 

Diplococcus  flavus —  +  +                      + 

Micrococcus  pharyngis  siccus      .  +  +  + 

Pigmented  coccus      I —  +  +                       +                       + 

II.       .-""•'." ...  :   .'',  .  +  +                       + 

"         III.       .      .      .      .      .  +  + 

Micrococcus  cinereus4 —  — 

It  will  be  seen  that  the  meningococcus  produces  acid  in  dextrose 
and  maltose.  A  differentiation  between  the  gonococcus,  Micrococcus 
catarrhalis  and  the  meningococcus  can  frequently  be  made  by  their 
growths  upon  cultural  media.  The  gonococcus  grows  poorly  or  not  at 
all  upon  blood  serum  (Loffler's),  the  meningococcus  grows  with  mod- 
erate rapidity  upon  it,  and  Micrococcus  catarrhalis  grows  even  upon 
plain  agar. 

The  final  diagnosis  of  the  meningococcus  depends  upon  its  agglu- 
tination with  specific  sera.  Positive  agglutination  will  take  place  in 
dilutions  of  1  to  500,  even  in  1  to  2000.  Kutscher5  has  isolated  strains 
of  the  organism  which  failed  to  agglutinate  (macroscopic  method)  at 
37°  C.,  but  agglutinated  typically  at  55°  C.  This  should  be  tried  in 
doubtful  cases. 

Serological  Diagnosis. — Bettencourt6  and  Franca,7  von  Lingelsheim, 
Elser  and  Huntoon8  and  others  have  shown  that  the  sera  of  convales- 
cent cases  of  cerebrospinal  meningitis  very  frequently  exhibit  specific 
agglutinins  for  the  meningococcus.  Of  593  tests,  von  Lingelsheim 
found  24.1  per  cent,  positive  during  the  first  five  days  of  the  disease, 
56.7  per  cent,  positive  from  the  sixth  to  the  tenth  day.  Normal  sera 
did  not  agglutinate  with  the  organism  in  dilutions  greater  than  1  to 
25;  the  sera  of  patients  agglutinated  in  dilutions  as  high  as  1  to  200. 
Elser  and  Huntoon  have  obtained  agglutination  in  dilutions  as  high 
as  1  to  400. 

The  method  of  complement-fixation  has  not  been  satisfactory  in 
the  diagnosis  of  cerebrospinal  meningitis.9 

1  +   =  Gram-positive  2  +  =  acid  produced 

-  =  Gram-negative  -  =  no  acid  produced. 

3  Jaeger's  meningococcus.  4  Micrococcus  catarrhalis? 

5  Kolle  and  Wassermann,  Handb.  d.  path.  Mikroorganismen,  I.  Erganzbd.,  1907,  518. 

6  Zeit.  f.  Hyg.,  1904,  xlvi,  463.  7  Klin.  Jahrb.,  1906,  xv,  Heft  2. 

8  Loc.  cit. 

9  Von  Lingelsheim  XIV  Cong,  for  Demog.  and  Hyg.,  Berlin,  September,  1907. 


300  THE  MENINGOCOCCUS—GONOCOCCUS  GROUP 

Dissemination  and  Prophylaxis.  —The  disease  is  usually  more  fre- 
quent in  children  and  young  adults,  usually  in  the  winter  and  spring 
months.  Frequently  a  nasal  inflammation  is  prevalent  before  the 
disease  begins  to  spread.  The  disease  spreads  by  contact;  as  the  organ- 
isms die  out  rapidly  away  from  the  human  body.  Many  cases  do  not 
progress  beyond  the  stage  of  nasal  pharyngitis  and  sore  throat,  and 
it  is  probable  that  these  cases  are  potentially  carriers.  According 
to  Bruns  and  Hohn,1  there  may  be  from  ten  to  twenty  times  as  many 
carriers  as  cases.  The  disease  is  very  likely  to  occur  in  barracks  and 
boarding  houses.  Many  people  may  be  exposed  to  infection  but 
comparatively  few  acquire  the  disease,  suggesting  a  rather  high 
natural  resistance  to  the  organism.  The  meningococcus  may  remain 
for  months  in  the  nasal  passages  of  carriers,  although  ordinarily 
they  remain  less  than  a  week. 

Ward  attendants  should  be  segregated  and  quarantined,  and  nasal 
sprays  used  on  the  patients  and  attendants.  It  is  quite  probable  that 
infected  handkerchiefs  or  inhalation  of  infectious  droplets  are  impor- 
tant in  spreading  the  organism.  It  should  be  treated  like  any  other 
acute  infectious  disease  of  the  respiratory  tract. 

Parameningococcus. — In  a  critical  discussion  of  the  treatment  of 
epidemic  cerebrospinal  meningitis  with  a  specific  antimeningococcus 
serum,  Flexner2  had  directed  attention  to  a  relatively  small  group  of 
cases  which  either  failed  to  respond  favorably  to  the  serum,  or  reacted 
for  a  short  time  and  later  failed  to  improve.  The  spinal  fluid  of  these 
cases  contained  organisms  microscopically  indistinguishable  from 
typical  meningococci.  It  was  assumed  tentatively  that  there  might 
be  two  types  of  meningococcus,  one  of  which  was  naturally  "serum- 
fast,"  the  other  acquired  "  serum-fastness"  during  the  course  of  the 
treatment  with  the  serum.  Dopter3  has  described  an  organism — the 
parameningococcus — apparently  identical  with  the  typical  meningo- 
coccus in  its  morphological  and  cultural  characteristics,  but  specifi- 
cally different  in  its  serological  reactions.  The  parameningococcus, 
like  the  meningococcus,  has  been  isolated  from  the  nasal  and  oral 
cavities  of  man,  and,  in  a  few  cases,  from  the  blood  stream  and  the 
meninges  as  well.  The  clinical  manifestations  incited  by  the  para- 
meningococcus are  indistinguishable  from  those  of  epidemic  cerebro- 
spinal meningitis,  but  they  fail  to  respond  favorably  to  the  adminis- 
tration of  meningococcus  serum.  Dopter4  has  prepared  a  specific 

1  Loc.  cit.  2  Jour.  Exp.  Med.,  1913,  xvii,  553. 

3  Compt.  rend.,  Soc.  de  Biol.,  1909,  Ixvii,  74.         4  Semaine  m6d.,  1912,  xxxii,  298. 


THE  GONOCOCCUS  GROUP  301 

• 

parameningococcic  serum  which  is  stated  to  have  effected  rapid 
improvement  in  the  few  cases  of  parameningococcus  infection  in  which 
it  was  tried.  These  cases  failed  to  respond  to  injections  of  meningo- 
coccus.  serum. 

Wollstein1  has  made  careful  comparative  studies  of  the  morpholog- 
ical, cultural  and  serological  reactions  exhibited  by  a  series  of  meningo- 
cocci  and  parameningococci;  her  conclusions,  which  follow,  summarize 
the  available  information  of  the  relationship  between  these  two 
organisms : 

'"The  parameningococci  of  Dopter  are  culturally  indistinguishable 
from  true  or  normal  meningococci,  but  serologically  they  exhibit 
differences  as  regards  agglutination,  opsonization,  and  complement 
deviation. 

"Because  of  the  variations  and  irregularities  of  serum  reactions 
existing  among  otherwise  normal  strains  of  meningococci,  it  does  not 
seem  either  possible  or  desirable  to  separate  the  parameningococci 
into  a  strictly  definite  class.  It  appears  desirable  to  consider  them 
as  constituting  a  special  strain  among  meningococci,  not,  however, 
wholly  consistent  in  itself. 

The  distinctions  in  serum  reactions  between  normal  and  paramen- 
ingococci are  supported  by  the  differences  in  protective  effects  of  the 
monovalent  immune  sera  upon  infection  in  guinea-pigs  and  monkeys. 

"  It  is  therefore  concluded  that  it  is  highly  desirable  to  employ  strains 
of  pararneningococcus  in  the  preparation  of  the  usual  polyvalent 
antimeningococcus  serum.  It  remains  to  be  determined  where  it  is 
better  to  employ  the  parameningococci  along  with  normal  meningo- 
cocci in  immunizing  horses,  or  to  employ  normal  and  para  strains 
separately 'in  the  immunization  process  and  to  combine  afterward,  in 
certain  proportions,  the  sera  from  the  two  kinds  of  immunized  horses." 

THE   GONOCOCCUS   GROUP. 

Micrococcus  Gonorrheas. — Synonyms. — Diplococcus  gonorrhese,  gon- 
ococcus. 

Historical. — The  gonococcus  was  first  observed  by  Neisser2  in  puru- 
lent urethral  and  vaginal  discharges.  Some  years  later  Bumm3  grew 
the  organism  in  pure  culture  upon  coagulated  human  blood  serum  and 
reproduced  acute  gonorrhea  in  men  by  urethral  injections. 

1  Jour.  Exp.  Med.,  1914,  xx,  201.  2  Cent.  f.  d.  med.  Wise.,  1879,  No.  28. 

3  Die  Mickroorganismen  des  gonorrhoischen  Schleimhauterkrankungen  Gonococcus, 
Neisser,  Wiesbaden,  1885,  No.  28. 


302 


THE  MENINGOCOCCUS—GONOCOCCUS  GROUP 


Morphology. — The  gonococcus  occurs  typically  as  a  diplococcus,  the 
proximated  surfaces  of  pairs  of  cocci  being  flattened  and  elongated; 
they  resemble  coffee  beans  in  shape.  The  longer  diameter  measures 
about  1.5  microns,  the  shorter  diameter  about  0.8  micron.  The 
polymorphonuclear  leukocytes  of  pus  from  cases  of  acute  gonorrhea 
usually  contain  from  one  to  several  pairs  of  gonococci  which  are  within 
the  cytoplasm  of  the  leukocyte  but  rarely  or  never  within  the  nuclei. 
The  organisms  are  also  found  within  desquamated  epithelial  cells  and 
occur  free  in  pus  as  well.  Gonococci  are  less  numerous  in  the  subacute 
and  chronic  stages  of  the  disease,  and  they  occur  chiefly  extracellu- 


FIG.  41. — Gonococcus  smear  of  pus  from  acute  case.    Methylene  blue  stain.    (Warden.) 

larly,  with  occasional  pairs  or  clusters  of  gonococci  in  epithelial  cells, 
less  commonly  in  polymorphonuclear  leukocytes.  The  organisms 
undergo  degeneration  rapidly,  and  even  in  pus  from  the  more  acute 
cases  many  large  faintly  staining  cocci  are  found  in  association  with 
those  which  are  more  typical  in  morphology  and  staining.  In  the 
chronic  stage  of  the  disease  degenerated  forms  are  very  common. 

The  gonococcus  is  non-motile,  and  possesses  no  flagella;  it  forms  no 
spores  and  capsules  have  not  been  detected.  It  stains  with  ordinary 
anilin  dyes,  but  with  some  difficulty.  It  is  Gram-negative. 

Isolation  and  Culture. — The  organism  does  not  grow  upon  ordinary 
media;  for  the  first  growths  outside  the  human  body  media  con- 
taining uncoagulated  protein,  preferably  that  of  human  origin,  is 


THE  GONOCOCCUS  GROUP  303 

required.  Agar1  smeared  with  sterile  defibrinated  blood,2  or  agar  mixed 
with  hydrocele  or  ascitic  fluid  (one  part  fluid,  two  parts  agar)  furnishes 
a  satisfactory  nutrient  substrate.  Pus  from  acute  cases  (after  pre- 
liminary cleaning  and  sterilization  of  the  external  genitalia)  spread 
upon  one  of  the  media  described  above,  should  exhibit  colonies  after 
twenty-four  hours'  incubation  at  37°  C.  The  colonies  are  minute, 
clear  and  colorless;  they  resemble  small  dewdrops  and  exhibit  a  ten- 
dency to  coalesce.  Organisms  stained  from  these  colonies  remain 
typical  in  morphology  only  for  one  or  two  days.  Very  soon  degen- 
eration (autolysis)  commences,  and  in  a  very  short  time  the  organisms 
are  dead3  and  partially  dissolved.  Secondary  growths  may  be  obtained 
from  colonies,  provided  the  inoculations  are  made  within  twenty-four 
to  forty-eight  hours  from  the  time  of  plating.  Ascitic  broth  is  an 
especially  favorable  medium  for  this  purpose. 

The  gonococcus  is  markedly  aerobic;  little  or  no  growth  occurs  in 
media  from  which  oxygen  is  excluded.  The  temperature  limits  are 
very  restricted;  growth  ceases  below  30°  C.  and  above  40°  to  42°  C. 
The  optimum  temperature  is  37°  C.  The  organism  is  extremely 
sensitive  to  desiccation,  and  cultures  die  spontaneously  within  six 
to  eight  days.  Repeated  transfers  of  the  cocci  at  intervals  of  two  to 
three  days  will  prolong  the  life  of  the  culture  almost  indefinitely,  pro- 
vided they  are  maintained  at  37°  C.  The  organisms  are  very  readily 
killed  (outside  the  body)  by  the^usual  disinfectants.  Gonococci  in 
the  urethra  can  not  be  killed  readily  by  chemical  disinfectants;  the 
organisms  penetrate  rather  deeply  into  the  walls  and  the  disinfectant 
can  not  reach  them  in  sufficient  concentration  to  be  effective.  This 
is  particularly  true  of  the  sub  acute  and  chronic  stages  of  the  disease. 

Products  of  Growth. — No  enzymes  have  been  detected  in  cultures  of 
gonococci.  Culturally  the  organism  is  inert;  no  development  occurs 
in  ordinary  media.  Acid  is  produced  in  dextrose-ascitic  broth,  but  no 
other  sugars  are  fermented.  (See  page  299  for  comparison  of  cultural 
characters  of  gonococcus  and  similar  Gram-negative  diplococci.) 

Toxins. — No  soluble  (exo-)  toxin  has  been  demonstrated  in  cultures 
of  gonococci. 

Finger,  Ghon  and  Schlagenhaufer,4  Nicolaysen,5  Wassermann6  and 
de  Christmas7  have  shown  that  the  cell  substance  itself  is  toxic. 

1  Glycerin  agar  is  better  than  ordinary  agar  for  this  purpose. 

2  The  blood  agar  should  be  heated  to  56°  C.  for  thirty  minutes  to  destroy  its  bacteri- 
cidal properties  before  use. 

3  Warden,  Jour.  Infec.  Dis.,  1913,  xii,  93. 

4  Arch.  f.  Derm.  u.  Syph.,  1894,  xxviii,  Nos.  1  and  2;    Cent.  f.  Bakt.,  1894,  xvi,  350. 

5  Cent.  f.  Bakt.,  1897,  xxii,  305.  6  Zeit.  f.  Hyg.,  1898,  xxvii,  307. 
7  Ann.  Inst.  Past.,  1900,  349. 


304  THE  MENINGOCOCCUS— GONOCOCCUS  GROUP 

De  Christmas  has  shown  that  the  poisonous  substance  (endotoxin) 
diffuses  readily  into  the  culture  medium,  probably  because  of  the 
rapid  autolysis  which  is  a  noteworthy  feature  of  the  organism.  The 
endotoxin  is  fairly  resistant  to  heat;  a  brief  exposure  to  120°  C.  fails 
to  entirely  destroy  its  potency. 

Pathogenesis. — Experimental. — Bumm1  and  Finger,  Ghon  and 
Schlagenhaufer2  have  reproduced  typical  urethritis  in  man  with 
pure  cultures  of  the  gonococcus.  The  latter  successfully  infected  the 
urethras  of  six  healthy  men  with  the  organism  (serum  agar  culture). 
The  incubation  period  was  from  two  to  three  days,  and  the  clinical 
picture  was  typical  in  each  instance.  The  organism  was  recovered 
in  pure  culture  from  each  patient. 

Animal. — Laboratory  animals  are  not  susceptible  to  urethral 
infection  with  the  gonococcus.  Intraperitoneal  injections  of  cultures 
into 'white  mice  produce  a  purulent  peritonitis,  but  there  is  little 
evidence  that  the  organisms  multiply  there.  Acute  joint  inflammations 
with  purulent  exudation  follows  the  inoculation  of  the  cocci  into  the 
joints  of  rabbits,  and  purulent  conjunctivitis  can  be  produced  in 
young  rabbits  by  rubbing  gonococci  on  the  conjunctiva.  There  is  no 
evidence  that  the  organisms  multiply  in  these  sites ;  the  reverse  appears 
to  be  the  case  for  the  cocci  disappear  rather  rapidly.  The  endotoxins 
are  responsible  for  the  local  reactions. 

Human. — Man  is  very  susceptible  to  infection  with  the  gonococcus. 
The  usual  portals  of  entry  are  the  mucous  membranes  of  the  urethra, 
vagina,  and  the  conjunctiva.  The  urethral  mucous  membrane  is 
particularly  susceptible  and  it  is  commonly  the  primary  site  of  inva- 
sion. The  uterine  mucosa  and  adnexa  are  also  readily  infected  in 
adults;  in  young  children  the  cervix  is  closed  and  infection  of  the 
uterus  by  continuity  of  growth  from  the  vagina  is  rare  in  them,  but 
vulvovaginitis  is  common,  especially  in  hospital  wards  where  infec- 
tion is  readily  transmitted  by  thermometers,  hands  of  ward  attendants, 
and  by  direct  contact. 

The  initial  development  of  the  organisms  is  upon  the  surface  of  the 
mucosa,  then  they  penetrate  to  the  deeper  layers,  infecting  the  pros- 
tate, and  by  continuity  the  epididymis  in  the  male.  Infection  may 
spread  from  the  vagina  to  the  uterus  in  the  female,  then  by  continuity 
of  growth  to  the  Fallopian  tubes,  the  ovaries,  and  the  peritoneum, 
causing  endometritis,  salpingitis,  oophoritis,  and  peritonitis.  Sterility 
is  usually  the  result.  Cystitis  and  arthritis  are  not  uncommon  sequelse 

1  Loc.  cit.  2  Loc.  cit 


THE  GONOCOCCUS  GROUP  305 

of  infection  with  the  gonococcus,  and  the  mucosa  of  the  rectum  is 
occasionally  involved.  Serous  surfaces  are  rarely  involved.  Occa- 
sionally a  generalized  invasion  takes  place  frequently  resulting  in 
septicemia  with  endocarditis.  Ophthalmia  neonatorum  is  a  particu- 
larly common  infection  of  the  newborn  of  infected  mothers.  The 
conjunctive  become  contaminated  with  gonococci  as  the  child  passes 
through  the  vagina.  A  large  percentage  of  the  blind  have  lost  their 
eyesight  in  this  manner  at  birth.  The  instillation  of  silver  prepara- 
tions, required  by  law  in  many  States,  has  greatly  reduced  this  form 
of  infection. 

Immunity. — Man  exhibits  little  or  no  resistance  to  infection  with 
the  gonococcus  and  the  mucous  membranes  may  actually  be  more 
susceptible  to  reinfection  than  they  were  originally.1  In  the  chronic 
cases,  where  the  organisms  lie  dormant  for  months,  even  years,  the 
tissues  appear  to  be  somewhat  less  suited  for  growth  of  the  organisms, 
but  the  patient  can  infect  others  even  at  this  stage  of  the  disease. 
Various  attempts  to  prepare  sera  for  curative  purposes  have  not  been 
generally  successful,  although  Rogers2  has  reported  cures  in  cases  of 
gonorrheal  rheumatism  and  chronic  gonorrheal  urethritis  by  the 
injection  of  the  serum  prepared  by  Torrey.3 

Vaccines  have  been  used  with  variable  success.  The  injection  of  an 
autogenous  vaccine,  containing  from  five  to  ten  million  gonococci 
from  a  twenty-four-hour  ascitic  fluid  agar  culture,  appears  to  give  the 
best  clinical  results.  Probably  the  extremely  rapid  autolysis  of  the 
gonococci  plays  a  prominent  part  in  the  ineffectual  attempts  to  induce 
improvement  by  the  use  of  vaccines.4 

Bacteriological  Diagnosis. — (a)  Microscopical. — Pus  from  the  urethra 
of  acute  cases  of  gonorrhea  should  be  dropped  upon  a  cover  glass  or 
slide  and  spread  by  gently  pressing  a  second  cover  glass  or  slide 
upon  the  first,  then  sliding  them  apart.  By  so  doing  the  organisms 
remain  in  the  polymorphonuclear  leukocytes  and  epithelial  cells,  a 
very  important  diagnostic  point.  A  Gram  stain  and  a  methylene- 
blue  stain  should  be  made.  The  former  reveals  intracellular  and 
intercellular  bean-shaped  diplococci  which  are  Gram-negative.  Occa- 
sionally leukocytes  contain  as  many  as  twenty  pairs  of  the  cocci. 
Dilute  methylene  blue  1  to  10  (Loffler's)  usually  stains  gonococci 
intensely;  the  remainder  of  the  cellular  elements  are  faintly  colored. 
The  morphology  of  the  organisms  is  clearly  shown  by  this  procedure. 

1  It  is  uncommon,  however,  to  find  auto  eye  infections  from  venereal  lesions;  even 
in  cases  of  gonorrheal  vulvovaginitis  the  eyes  are  rarely  infected  with  the  gonococcus. 

2  Am.  Jour.  Med.  Sc.,  1906,  xlvi,  No.  4. 

a  Ibid.  4  Lespinasse;  Illinois  Med.  Jour.,  April,  1912. 

20 


306  THE  MENINGOCOCCUS—GONOCOCCUS  GROUP 

In  chronic  cases  the  discharge  is  scanty  and  it  is  better  to  receive 
the  morning  urine  in  a  sedimentation  glass  containing  a  crystal  or  two 
of  thymol.  After  a  short  time  threads  of  mucus  separate  out;  these 
should  be  removed  with  a  capillary  pipette  and  examined  as  above. 
The  pus  from  old  cases  of  gonorrhea  frequently  contains  but  few  gono- 
cocci,  which  are  difficult  to  find.  It  has  been  found  that  the  local 
injection  of  silver  nitrate  (properly  diluted)  will  usually  cause  an 
elimination  of  pus  which  frequently  contains  the  organisms  in  some- 
what larger  numbers.  Drinking  beer  is  said  to  produce  the  same 
result.  Vaginal  smears  may  be  obtained  from  swabs  which  are  intro- 
duced into  the  vagina,  or  by  means  of  long  pipettes  with  rounded  ends, 
containing  a  few  drops  of  1  to  1000  mercuric  chloride,  which  are 
expressed  and  drawn  up  into  the  pipette  several  times  deep  in  the 
vagina.  The  material  thus  removed  is  stained  in  the  usual  manner. 
Smears  from  the  conjunctiva  should  be  diagnosed  very  conservatively; 
Micrococcus  catarrhalis  and  other  Gram- negative  diplococci  which 
may  occur  within  polymorphonuclear  leukocytes  are  occasionally 
associated  with  an  inflamed  conjunctiva.  The  clinical  picture  should 
be  considered  in  making  the  final  diagnosis  in  such  cases,  and  whenever 
possible  cultures  should  be  made  to  confirm  the  results. 

(b)  Cultural. — Cultures  of  the  gonococcus  are  best  obtained  early 
in  the  disease,  when  secondary  infection  with  staphylococci  or  other 
organisms  has  not  taken  place.  The  external  genitalia  should  be 
carefully  cleaned  as  for  a  surgical  operation,  and  pus  collected  on  a 
sterile  swab  which  is  rubbed  over  the  surface  of  blood-  or  ascitic  agar. 
The  isolation  of  gonococci  from  pus  of  the  subacute  and  chronic  stages 
of  the  disease  is  extremely  difficult;  indeed,  it  is  practically  a  matter 
of  chance  if  pure  cultures  are  obtained  at  this  time.  Vaginal  cultures 
may  be  obtained  upon  sterile  swabs  which  are  inoculated  in  the  same 
manner. 

The  gonococcus  does  not  grow  upon  ordinary  media,  not  even 
Loffler's  blood  serum,  which  distinguishes  it  from  the  meningococcus 
and  from  other  Gram-negative  cocci,  including  Micrococcus  catar- 
rhalis. (For  fermentation  reaction  of  the  gonococcus,  see  page  299). 

Serological  Diagnosis. — Agglutinins. — The  diagnosis  of  gonorrhea 
by  agglutination  of  the  gonococcus  with  the  serum  of  the  patient  has 
not  been  successful.1 

1  Torrey  (Journ.  Med.  Res.,  1908,  xx,  771)  has  isolated  ten  strains  of  gonococcus 
identical  morphologically  and  culturally,  but  distinct  serologically.  This  may  explain 
in  part  the  irregularity  of  the  reaction  of  agglutination  provided  but  a  single  strain  of 
organism  is  used. 


THE  GONOCOCCUS  GROUP  307 

Complement-fixation  Reaction. — Diagnosis  of  gonococcus  infection 
by  the  method  of  complement  fixation  has  been  shown  to  be  of  con- 
siderable value,  particularly  in  the  more  chronic  cases,  provided  an 
homologous  strain  of  the  organism  is  used  for  the  antigen.  A  mixed 
antigen  composed  of  several  strains  is  frequently  employed  in  prac- 
tice.1 Much  additional  work  is  required,  however,  to  determine  the 
limits  of  variability  of  the  various  strains  of  the  organism  before  the 
method  is  placed  upon  a  thoroughly  satisfactory  basis  for  routine  work. 

Shattuck  and  Whittemore2  have  prepared  concentrated  polyvalent 
glycerin  extracts  and  autolysates  of  gonococci  to  test  the  value  of  the 
skin  reactions  in  gonococcus  infections.  The  tests  were  made  intra- 
dermally  and  by  the  von  Pirquet  method.  Their  results  were 
in) satisfactory  diagnostically. 


FIG.  42. — Micrococcus  catarrhalis  and  staphylococcus. 

The  medicolegal  aspects  of  gonorrheal  infections  make  it  incumbent 
upon  the  examiner  to  be  very  cautious  in  diagnosing  the  organism. 

Dissemination  and  Prophylaxis. — The  common  towel  has  in  the  past 
been  responsible  for  many  cases  of  gonorrheal  ophthalmia,  but  laws 
forbidding  its  use  have  largely  removed  this  danger.  It  is  certain 
that  ordinary  care  will  prevent  infection  of  the  innocent  with  th^ 
organism.  Ophthalmia  neonatorum  is  prevented  by  the  instillation 
of  silver  salts  in  the  manner  indicated  above. 

Micrococcus  Catarrtiklis. — Historical. — Micrococcus  catarrhalis  ap- 
pears to  have  been  described  first  by  Seifert3  and  by  Kirchner;4  the 

1  Lespinasse  and  Wolff,  Illinois  Med.  Jour.,  January,   1913.     Torrey's  ten  strains 
should  be  used  in  preparing  the  gonococcus  antigen. 

2  Boston  Med.  and  Surg.  Jour.,  1913,  xlxix,  373. 

3  Volkmann's  Sammlung  klinischer  Vortrage,  No.  240. 
«  Zeit.  f.  Hyg.,  1890,  ix,  528. 


308  THE  MENINGOCOCCUS—GONOCOCCUS  GROUP 

name  first  appears  in  Die  Mikroorganismen  (Fliigge),  3d  edition,  in 
1896,  credited  to  R.  Pfeifl'er. 

Morphology. — Micrococcus  catarrhalis  occurs  typically  as  a  diplo- 
coccus  with  the  apposed  surfaces  of  adjacent  cocci  flattened  and 
somewhat  elongated.  It  measures  about  one  micron  in  diameter. 
Occasionally  the  organisms  are  arranged  in  tetrads,  particularly  in 
young,  active  cultures  in  artificial  media;  in  older  cultures  a  tendency 
toward  short  chain  formation  is  frequently  observed.  Degenerated 
cocci  occur  in  older  cultures.  In  sputum,  bronchial  secretions  and 
other  material  from  inflammation  of  the  upper  respiratory  tract,  in 
which  Micrococcus  catarrhalis  is  a  primary  or  accessory  factor,  the 
organisms  occur  both  within  and  without  the  pus  cells.  In  the  acute 
stages  they  are  usually  extracellular.1  The  organism  is  non-motile, 
and  it  has  no  flagella.  It  forms  neither  spores  nor  capsules.  It  colors 
readily  with  ordinary  anilin  .dyes,  some  cells  more  intensely  than  their 
fellows,  and  it  is  Gram-negative. 

Isolation  and  Culture. — The  organism  grows  with  moderate  vigor 
upon  agar;  after  twenty-four  hours'  incubation  the  colonies  are 
small,  translucent  and  gray.  After  three  to  four  days  the  colonies 
are  larger  with  an  opaque  centre,  the  periphery  being  translucent. 
Old  colonies  tend  to  become  somewhat  brownish.  Development  is 
more  vigorous  in  media  containing  blood,  blood  serum,  or  ascitic 
fluid.  Hemolysis  of  the  blood  does  not  occur.  The  growth  in  gelatin 
is  slow,  and  usually  feeble.  A  slight  turbidity  develops  in  broth. 
Moderate  development  occurs  in  milk.  Micrococcus  catarrhalis 
grows  best  at  37°  C.;  restricted  development  takes  place  at  16°  C.; 
no  growth  can  be  detected  at  43°  C. 

Products  of  Growth. — The  organism  is  culturally  inert.  It  does  not 
produce  any  demonstrable  proteolytic  enzymes,  and  it  produces  no 
acid  in  any  sugar.  No  toxic  products  are  known.  Filtrates  of  broth 
cultures  have  no  apparent  action  upon  white  mice.  No  pathogenesis 
for  laboratory  animals  has  been  detected. 

Human  Pathogenesis. — Micrococcus  catarrhalis  has  occasionally  been 
reported  as  a  causative  factor  in  catarrhal  inflammations  of  the  upper 
respiratory  tract,  and  even  in  atypical  pneum6nia2  and  in  bronchitis.3 
Ordinarily  it  is  an  opportunist  found  in  the  upper  respiratory  tract. 

Bacteriological  Diagnosis. — The  organism  is  of  importance  chiefly 
through  its  striking  resemblance  to  the  meningococcus  and  the  gono- 

1  Ghon,  Pfeiffer  and  Sederl,  Zeit.  f.  klin.  Med.,  1902,  xliv,  262. 

2  Bernheim,  Deut.  med.  Wchnschr.,  1900.       3  Ritchie,  Jour.  Path,  and  Bact.,  1900 


THE  GONOCOCCUS  GROUP  309 

coccus.  It  differs  from  these  diplococci  both  in  its  relatively  luxuriant 
growth  upon  artificial  media  and  its  ability  to  grow  at  room  tem- 
perature. It  resembles  them  in  its  intracellular  disposition  and  in  its 
staining  reactions. 

Droplet  infection  and  transmission  by  contact  are  possible  means 
of  dissemination,  and  appropriate  precautions  should  be  taken  to 
prevent  this. 


CHAPTER  XV. 

MICROCOCCUS  MELITENSIS. 

Historical. — The  organism  was  discovered  by  Bruce.1 

Morphology. — Micrococcus  melitensis  is  a  very  small  oval  coccus, 
occurring  singly  or  in  pairs,  rarely  in  short  chains;  the  individual 
cells  measure  about  0.3  to  0.4  micron  in  diameter.  Some  observers 
declare  the  organism  to  be  a  short  bacillus,  a  view  which  is  perhaps 
based  upon  its  appearance  in  old  cultures,  where  various  involution 
forms  are  readily  observed.  The  coccus  form  almost  invariably  pre- 
dominates in  fresh  material.  The  organism  is  non-motile,  possesses 
no  flagella  and  forms  no  capsule.  Spore  formation  has  never  been 
observed.  It  stains  readily*  with  ordinary  anilin  dyes,  and  is  Gram- 
negative. 

Isolation  and  Culture. — One  of  the  noteworthy  cultural  characters 
of  Micrococcus  melitensis  is  its  slow  growth  on  artificial  media,  even 
at  37°  C.  Suspected  material,  either  blood,  urine,  milk,  or  material 
from  splenic  puncture,  should  be  spread  upon  the  surface  of  slightly 
acid  agar  and  examined  after  three  or  four  days'  incubation  for  very 
minute  white  colonies  which  have  a  darker  center.  The  organism 
grows  slowly  in  gelatin,  without  producing  liquefaction,  and  it  pro- 
duces a  slight  turbidity  in  broth.  Milk  appears  to  be  a  good  medium 
for  its  development,  goats'  milk  being  better  than  cows'  milk  for  this 
purpose. 

The  coccus  is  aerobic,  facultatively  anaerobic.  The  minimum  tem- 
perature of  growth  is  about  8°  C.,  the  optimum  37°  C.,  and  the 
maximum  about  44°  C.  Direct  sunlight  kills  it  in  a  few  hours;  an 
exposure  to  55°  C.  is  usually  fatal  within  an  hour;  1  per  cent,  carbolic 
acid  kills  it  in  ten  to  fifteen  minutes.2  It  resists  drying  in  the  cold 
and  in  the  dark  for  several  weeks. 

Products  of  Growth. — ^Micrococcus  melitensis  is  culturally  inert;3  it 
produces  no  proteolytic  enzymes  and  it  produces  neither  acid  nor  gas 
in  any  sugars.  Milk,  particularly  goats'  milk,  becomes  progressively 
alkaline  in  reaction.  No  toxins  have  been  demonstrated. 

1  Practitioner,  September,  1887,  xxxix,  161. 

2  Mohler  and  Eichhorn,  Bureau  of  Animal  Industry,  1911,  xxviii,  125. 

3  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Soc.,  1913,  xxxv,  1247. 


IMMUNITY  AND  IMMUNIZATION 


311 


Pathogenesis. — Animal. — Apes  are  susceptible  to  Micrococcus  meli- 
tensis;  the  subcutaneous  inoculation  of  cultures  of  the  organism 
leads  to  definite  clinical  symptoms  parallel  to  those  observed  in 
man.  The  disease  usually  runs  a  prolonged  course  and  is  often  fatal. 
Monkeys  are  somewliat  less  favorable  subjects  than  apes.  (Goats, 
sheep,  cattle,  and  horses  are  also  susceptible  to  infection,  although 
the  disease  is  rarely  generalized;  the  presence  of  the  virus  in  the  urine 
of  the  males,  the  milk  and  urine  of  the  females  of  these  species  is  the 
principal  indication  of  infection.)  The  incubation  period  is  from 
five  to  fourteen  days.  Eyre1  spates  that  rabbits  and  guinea-pigs  may 
be  infected,  but  not  rats  and  mice. 


FIG.  43.— Micrococcus  melitensis  and  staphylococcus.      X  1000.     (Kolle  and  Hetsch.) 

Milk  appears  to  be  the  chief  source  of  infection;  on  the  Island  of 
Malta,  where  Malta  fever  was  first  described,  fully  10  per  cent,  of 
the  female  goats  contained  the  organism  in  their  milk.  Monkeys 
readily  contracted  the  disease  by  drinking  this  milk.  The  urine  of 
both  male  and  female  goats  was  shown  to  be  infected  as  well. 

Immunity  and  Immunization. — The  blood  and  urine  of  infected  indi- 
viduals contain  the  virus  of  the  disease  and  specific  agglutinins  are 
present  in  the  blood  even  early  in  the  disease.  The  agglutinins  may 
persist  for  years  after  convalescence.  Dilutions  of  ^  to  joioo 
are  made  from  the  blood  serum  with  suitable  controls.  A  small  amount 
of  growth  from  a  three-day  agar  culture  of  the  organism  is  thoroughly 
emulsified  in  each  dilution  of  serum  and  in  the  controls;  the  emulsions 
are  incubated  at  37°  C.  for  two  hours,  then  placed  in  the  ice-box  for 
twenty-four  hours  before  the  readings  are  made.  A  control  with  a 

1  Kolle  u.  Wasserman,  Handb.  d.  Path.  Mikroorganismen,  I.  Erganzband. 


312  MICROCOCCUS  MELITENSIS 

non-specific  serum  (g$)  and  the  organism  should  be  made  at  the 
same  time  and  incubated  in  the  same  manner,  for  experience  has 
shown  that  the  serum  of  normal  individuals  may  agglutinate  Micro- 
coccus  melitensis  in  moderately  high  dilution.  Wright  has  immunized 
horses  with  repeated  injections  of  Micrococcus  melitensis.  The  blood 
serum  agglutinated  the  organism  in  high  dilution;  it  was  claimed  by 
'him  that  the  serum  possessed  curative  value,  the  chief  phenomena 
following  its  administration  being  a  fall  in  temperature  and  a  shorten- 
ing of  the  course  of  the  disease.  This  is  still  debatable. 

Bacteriological  Diagnosis. — A.  Blood,1  urine,  milk,  or  material  from 
splenic  puncture  is  plated  out  as  outlined  above.  The  organisms  are 
agglutinated  with  a  serum  of  high  potency. 

B.  The  blood  of  the  patient  should  be  examined  in  high  dilution 
(lUo)  f°r  specific  agglutinins. 

Dissemination  and  Prophylaxis. — The  organisms  leave  the  body 
through  the  milk  or  urine.  Pasteurization  of  the  milk  and  disinfection 
of  the  urine  of  infected  animals  is  the  best  prophylaxis.  It  should 
be  remembered  that  the  organisms  can  enter  the  body  through 
cutaneous  wounds. 

1  The  organisms  are  not  always  present  in  the  blood  of  patients  in  demonstrable 
numbers;  a  negative  culture  is  not  conclusive. 


CHAPTER  XVI. 

THE  ALCALIGENES— DYSENTERY— TYPHOID— PARA- 
TYPHOID GROUP. 

BACILLUS    ALCALIGENES. 

BACILLUS  alcaligenes  was  first  isolated  by  Petruschky1  from  the  feces 
of  a  patient  presenting  the  clinical  symptoms  of  typhoid  fever.  The 
serum  did  not  agglutinate  the  typhoid  bacillus  and  no  typhoid  bacilli 
were  recovered  from  the  blood  or  dejecta.  Several  similar  cases  are 
now  on  record  in  which  Bacillus  alcaligenes  has  been  isolated  both 
from  the  blood  stream  and  the  intestinal  contents;  the  sera  of  these 
cases  agglutinated  the  specific  organism  in  dilutions  of  1  to  50 
or  even  higher,  and  Bacillus  typhosus  was  not  found.  Bacillus 
alcaligenes  occurs  occasionally  in  acute  intestinal  disturbances  of 
young  children,  not  infrequently  in  association  with  organisms  of  the 
dysentery  and  paratyphoid  groups.2  Less  commonly  it  is  found  in 
the  dejecta  of  normal  children,  adults3  and  in  water. 

Morphology. — The  organism  both  in  size  and  shape  resembles  the 
typhoid  bacillus  very  closely.  It  is  actively  motile  and  has  peritrichic 
flagella.  It  does  not  form  spores,  and  so  far  as  is  known,  does  not 
exhibit  a  capsule.  Ordinary  anilin  dyes  color  it  readily  and  it  fails  to 
retain  the  Gram  stain. 

Isolation  and  Cultures. — The  organism  grows  readily  in  ordinary 
media.  On  agar  the  colonies  are  transparent,  colorless,  and  round, 
and  after  eighteen  hours'  incubation  at  37°  C.  attain  a  diameter  of 
from  1  to  3  mm.  The  organism  grows  with  moderate  luxuriance  on 
gelatin,  but  produces  no  liquefaction.  In  broth  theie  is  a  uniform 
clouding,  and  after  a  few  days  a  delicate  pellicle  usually  forms.  Bacillus 
alcaligenes  grows  fairly  readily  in  milk;  the  reaction  becomes  progres- 
sively alkaline.  In  sugars  no  acid  or  gas  is  developed. 

The  organism  is  aerobic,  facultatively  anaerobic.  The  minimum 
temperature  of  growth  is  about  6°  C.,  the  optimum  37°  C.,  and  the 

1  Cent.  f.  Bakt.,  1896,  xix,  187. 

2  Kendall,  Day  and  Bagg,  Boston  Med.  and  Surg.  Jour.,  1913,  clxix,  741. 

3  Ford,  Studies  from  the  Royal  Victoria  Hospital.  Montreal,  1903,  i,  No.  5. 


314 


THE  ALCALI&ENES— DYSENTERY— TYPHOI  I) 


maximum  about  44°  C.  The  resistance  of  Bacillus  alcaligenes  to 
physical  and  chemical  reagents  is  similar  to  that  of  the  typhoid  bacillus. 
Products  of  Growth. — Bacillus  alcaligenes  is  characterized  culturally 
by  its  inertness.  Neither  acid  nor  gas  is  produced  from  any  known 
sugar.  A  moderate  amount  of  proteolysis  similar  in  degree  to  that 
of  the  typhoid  bacillus  in  sugar-free  broth  is  characteristic  of  the 
development  of  this  organism  in  all  the  ordinary  media.1  Milk  is 
not  coagulated  nor  peptonized,  but  a  progressive  alkalinity  develops, 
associated  with  the  liberation  of  small  amounts  of  ammonia.2  No 
enzymes  have  been  detected,  and  no  toxins  have  been  demonstrated 
in  cultures  of  the  organism. 


FIG.  44.     Bacillus  alcaligenes;  bouillon  culture.      X  1000. 

Pathogenesis. — The  comparatively  few  cases  of  infection  with 
Bacillus  alcaligenes  have  not  been  studied  in  sufficient  detail  to  throw 
any  light  upon  the  character  of  the  lesions  produced  by  the  organism. 
The  disease  resembles  typhoid  fever  clinically,  and  it  is  possible  that 
in  the  past  occasional  typhoidal  fevers  have  been  incorrectly  diagnosed. 
Animal  experimentation  has  been  uniformly  negative. 

Immunity. — Nothing  definite  is  known  of  the  immunological  rela- 
tions of  Bacillus  alcaligenes.  Specific  agglutinins  have  been  demon- 
strated in  a  few  instances  where  infection  with  the  organism  has  been 
confirmed  bacteriologically. 

Bacteriological  Diagnosis. — The  organism  may  be  isolated  occasion- 
ally from  the  blood;  ordinarily,  however,  the  diagnosis  is  made  by 
the  isolation  of  the  bacilli  from  the  feces.  Upon  the  Endo  medium  the 
organism  grows  precisely  like  the  typhoid  bacillus.  It  is  readily  dif- 

1  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Assn.,  1913,  xxxv,  1216. 

2  Ibid.,  1914,  xxxvi,  1940. 


THE  GROUP  OF   THE  DYSENTERY  BACILLI  315 

ferentiated  from  the  typhoid  bacillus  by  cultural  reactions,  Bacillus 
alcaligenes  forming  neither  acid  nor  gas  in  dextrose,  lactose,  saccharose, 
or  mannite.  It  does  not  liquefy  gelatin,  and  it  produces  a  permanent 
alkalinity  in  milk.  The  differential  cultural  reactions  are  shown  in 
the  table  (page  316). 

Dissemination  and  Prophylaxis. — Nothing  is  known  of  the  method  of 
dissemination  of  Bacillus  alcaligenes.  It  appears  to  be  an  organism 
whose  portal  of  entry  is  the  gastro-intestinal  tract.  Carriers  have 
never  been  satisfactorily  demonstrated.  Prophylaxis  is  precisely  the 
same  as  that  for  other  intestinal  organisms. 

THE   GROUP   OF   THE   DYSENTERY  BACILLI. 

The  term  dysentery  as  it  is  used  in  the  clinical  way  includes  at 
least  two  entirely  distinct  entities:  amebic  dysentery,  a  semi-acute 
or  chronic  infection  caused  by  an  ameba,  which  is  usually  restricted 
to  the  tropics  and  subtropics;  and  an  acute  type  caused  by  members 
of  the  dysentery  bacillus  group,  more  frequently  encountered  in 
temperate  zones.  The  latter  type  not  uncommonly  assumes  epidemic 
proportions,  but  occurs  sporadically  as  well.  Japan  has  suffered  greatly 
in  the  past  from  the  ravages  of  bacillary  dysentery.  Ogata  and 
Eldridge1  state  that  1,136,067  cases  with  257,289  deaths  occurred  in 
that  country  during  the  period  between  1878  and  1899  inclusive. 
The  mortality,  which  varied  markedly  from  year  to  year,  averaged 
22.6  per  cent,  of  all  cases.  The  disease  appears  to  be  rare  in  England, 
but  it  has  been  reported  in  Germany.2  The  Atlantic  seacoast  cities 
of  the  United  States  have  experienced  epidemics  of  the  disease,  but 
the  inland  cities  appear  to  have  been  relatively  free  from  it.  During 
inter-epidemic  years  mild,  atypical,  sporadic  cases  and  moderate 
numbers  of  bacilli  carriers  (both  of  the  Shiga  and  Flexner  types  of 
organisms)  have  been  discovered.3 

The  most  virulent  of  the  dysentery  bacilli  was  isolated  and  described 
by  Shiga4  during  the  great  epidemic  of  1897  to  1898  in  Japan.  Flexner5 
recovered  an  organism  which  he  believed  was  identical  with  the  Shiga 
bacillus  from  cases  of  dysentery  in  the  Philippines.  Later  studies  of 
this  organism  by  Martini  and  Lentz6  revealed  specific  differences  in 

1  Quoted  in  Public  Health  Reports,  1900,  xv,  1. 

2  Kruse,  Deut.  med.  Wchnschr.,  1900,  vol.  xxvi. 

3  Kendall,  Boston  Med.  and  Surg.  Jour.,  1913,  clxix,  754;    May  20,  1915.  • 

4  Cent.  f.  Bakt.,  1898,  xxiii,  599;    xxiv,  817,  870,  913. 
B  Ibid.,  1900,  xxviii,  625. 

6  Zeit.  f.  Hyg.,  1902,  xli,  540. 


316 


THE  ALCALIGENES— DYSENTERY— TYPHOID 


agglutinins  from  the  Shiga  bacillus,  and  Lentz1  showed  that  the  Shiga 
bacillus  did  not  ferment  mannite;  the  Flexner  bacillus  ferments  this 
alcohol  with  the  production  of  acid.  Later  intensive  studies  of  bacil- 
lary  dysentery  bacilli  by  Park  and  Dunham,  Hiss  and  Russell,  and 
others  confirmed  the  work  of  the  earlier  observers  and  added  several 
strains  to  the  group,  which  differ  from  the  Shiga  and  Flexner  strains 
both  with  respect  to  their  specific  agglutinating  powers  and  their 
cultural  reactions.  The  principal  cultural  reactions  of  the  more 
prominent  Gram-negative  intestinal  bacteria,  including  not  only  the 
pathogenic  organisms  but  the  habitually  parasitic  organisms  as  well, 
follow : 


O 

Motility. 

Dextrose. 

Lactose. 

03 

Mannite. 

Levulose. 

Galactose. 

Maltose. 

Gelatin 
liquefaction. 

d 
§ 

B.  alcaligenes    
B.  dysenterise  Shiga     .... 
B.  dysenterise  Flexner 
B.  dysenterise  Hiss-  Russell     . 
B.  dysenterise  Rosen    .... 
B.  pyogene^  fcetidus    .      . 
B.  typhosus  a-  .      .      .      .      .      . 
B.  typhosus  b  
B.  para  typhosus  alpha 
B.  paratyphosus  beta 
B.  Morgan  No.  1 

- 

+ 

+ 
+ 
+ 
+ 
+ 
+ 
-f- 

-h 
+' 
+ 
+ 
+ 
+ 
+ 
g 
g 

+ 

+ 

+ 
+ 

+ 
+ 
+ 
+ 
+ 
+ 
g 
g 

+ 
+ 
+ 
.  + 
+ 
+ 
g 
g 
? 

+ 

+ 
+ 
+ 
+ 
+ 
g 

g 
? 

+ 
+ 
+ 
+ 
+ 
g 

g 
? 

- 

=±= 
± 
± 
+ 
+ 
± 

+ 

+ 
± 

-4- 

B.  coli  a       
B.  coli  b       
B.  proteus   
B.  cloacae     

- 

+ 
+ 
+ 
+ 

g 
g 

g 
g 

g 

4 

g 

g 
g 
g 

g 
g 

g 

g 
g 
g 
g 

g 
g 
g 
g 

g 
g 
g 
g 

+ 

+ 

C1 

c 

p 
c/p 

Legend:  carbohydrate  solutions:      -  =  no  fermentation,  +  =  acid  produced,  g  =  gas 

produced. 

milk:     —  =  no  fermentation,  alkaline  reaction,  =*=    =  initial  acidity,  terminal 
alkalinity,   +  =  acid,  c  =  coagulation,  p  =  peptonization. 

Morphology. — The  morphology  of  the  members  of  the  dysentery 
group  of  bacilli  is  practically  identical;  they  are  medium-sized,  rod- 
shaped  organisms,  measuring  from  0.8  to  1  micron  in  diameter,  and 
from  1.5  to  3  microns  in  length.  They  have  rounded  ends  and  occur 
singly  or  in  pairs,  rarely  in  short  chains.  Frequently  elongated 
somewhat  irregular  involution  forms  are  found  in  old  broth  cultures. 
The  bacilli  are  non-motile  (except  the  "Rosen"  strain,  which  is  slug- 
gishly motile),  possess  no  flagella,  form  no  capsules  and  produce  no 
spores.  They  stain  fairly  readily  with  ordinary  anilin  dyes;  frequently, 


1  Zeit.  f.  Hyg.,  1902,  p.  559. 


THE  GROUP  OF  THE  DYSENTERY  BACILLI  317 

the  ends  of  the  organisms  stain  somewhat  more  heavily  than  the 
centre.    All  the  organisms  comprising  this  group  are  Gram-negative. 

Isolation  and  Culture. — The  dysentery  bacilli  grow  well  on  ordinary 
laboratory  media.  Colonies  on  agar,  after  eighteen  to  twenty-four 
hours'  incubation  at  the  body  temperature,  are  round,  transparent 
and  colorless;  frequently  they  attain  a  diameter  of  from  1  to  3  mm. 
The  colonies  are  indistinguishable  from  those  produced  by  bacilli  of 
the  typhoid  and  paratyphoid  groups.  There  is  moderate  growth 
along  the  line  of  inoculation  in  gelatin,  but  no  liquefaction.  In  broth 
after  eighteen  to  twenty-four  hours'  growth  a  uniform  turbidity 
develops,  somewhat  more  luxuriant  in  dextrose  than  in  plain  broth. 
After  several  days'  growth  in  plain  broth  a  delicate  pellicle  frequently 


FIG.  45. — Bacillus  dysenterise.     Shiga  type,  bouillon  culture.      X  1000.  . 

appears  on  the  surface  of  the  latter  medium.  In  milk  moderate  devel- 
opment takes  place  with  no  coagulation.  There  is 'an  initial  acidity 
followed  after  from  two  to  five  days  by  an  alkaline  reaction,  which 
increases  somewhat  in  intensity  with  the  age  of  the  culture.  On  potato 
the  growth  is  very  similar  to  that  of  the  typhoid  bacillus;  on  acid 
potato  the  growth  is  almost  invisible;  on  alkaline  potato  the  growth 
is  brownish  and  of  moderate  luxuriance. 

The  dysentery  bacilli  are  aerobic,  facultatively  anaerobic  bacilli 
whose  limits  are  approximate^fcthe  following;  minimum  temperature 
of  growth  8°  C.;  maximum  42°  to^°  C.;  optimum  37°  C. 

Cultures  of  dysentery  bacilli  varyWimewhat  in  their  resistance  to 
heat.  The  majority  of  cultures  are  killed  by  an  exposure  of  ten  min- 
utes at  65°  C.  Some  strains,  however,  are  only  killed  by  an  exposure 
of  ten  minutes  at  70°  C.  The  organisms  are  moderately  resistant  to 


318  THE  ALCALIGENES— DYSENTERY— TYPHOID 

cold.  Cultures  may  retain  their  viability  in  the  ice-box,  6°  to  10°  C., 
for  nearly  two  months.  In  sterile  water  the  organisms  at  ordinary 
temperatures  do  not  as  a  rule  survive  more  than  a  week.  Pfuhl1  has 
found  that  dysentery  bacilli  may  remain  alive  for  101  days  in  moist 
soil  protected  from  sunlight;  in  dry  soil  under  otherwise  the  same 
conditions  they  do  not  survive  more  than  thirty  days.  In  cheese  and 
in  butter  they  remain  alive  for  at  least  nine  days,  and  in  sterile  milk  for 
about  three  weeks.  Dried  on  linen,  they  also  survive  about  three  weeks. 

Products  of  Growth. — Chemical  Products. — Plain  broth  cultures  of 
Shiga  and  Flexner  bacilli  do  not  contain  indol  or  phenols,  even  after 
prolonged  incubation.  The  statements  with  reference  to  indol  produc- 
tion in  the  group,  however,  are  somewhat  conflicting,  particularly 
with  reference  to  the  Flexner  type  of  organism.  Morgan  and  others2 
have  stated  that  Flexner  bacilli  produce  indol;  on  the  other  hand, 
Kendall,  Bagg,  Day  and  Walker3  have  isolated  over  200  strains  of 
Flexner  bacilli  from  dysenteric  cases  and  have  found  almost  without 
exception  that  indol  is  not  formed.  These  strains  were  identified  by 
their  cultural  reactions  and  by  agglutination  with  specific  Flexner 
serum  of  high  potency.  Dopter4  has  found  that  strains  of  Flexner 
bacilli  obtained  from  different  sources,  which  were  identical  culturally 
and  agglutinated  the  same  with  specific  sera,  vary  in  indol  production 
some  producing  indol,  others  not  producing  it. 

Acid  Production  in  Carbohydrate  Media. — All  members  of  the  dysen- 
tery group  agree  in  two  important  characteristics:  they  do  not  form 
gas  in  carbohydrate  media,  and  form  acid  in  dextrose.  Lentz5  has 
called  attention  to  an  important  cultural  differentiation  of  the  Flexner 
and  Shiga  bacillus,  the  former  producing  acid  in  mannite,  the  latter 
not  fermenting  this  alcohol.  Further  study  has  shown  that  the  fer- 
mentation of  various  carbohydrates  is  important  in  the  recognition 
of  the  various  types.  The  fermentation  and  other  cultural  reactions 
of  members  of  the  dysentery  bacillus  group  are  shown  in  the  table  on 
page  316.  The  members  of  the  dysentery  group  produce  an  initial 
acidity  in  milk;  fermentation  of  the  small  amount  of  dextrose,  amount- 
ing to  about  0.1  per  cent.,  which  is  found  in  fresh  milk  (Theobald 
Smith6)  followed  by  an  .alkaline  reaction  (action  of  the  organisms 
upon  protein  when  the  utilizable  carbohydrate  is  exhausted).7 

i  Ztschr.  f.  Hyg.,  1902,  xl,  555.  2  Brit.  Med.  Jour.,  April  6,  1907,  908;  July  6,  16. 

3  Boston  Med.  and  Surg.  Jour.,   1911,  clxiv,  301;    1913,  clxix,  741,  753;    Jour.  Am. 
Chem.  Soc.,  1913,  xxxv,  1211. 

4  Les  Dysenteries,  Paris,  1909,  36.  6  Ztschr.  f.  Hyg.,  1902,  xli,  559. 

5  Boston  Jour.  Med.  Sci.,  1897,  ii,  236;    Jones,  Jour.  Inf.  Dis.,  1914,  xv,  357. 

7  See  Kendall,  Day  and  Walker  for  essential  analytical  details,  Jour.  Am.  Chem. 
Assn.,  1914,  xxxvi,  1940, 


THE  GROUP  OF  THE  DYSENTERY  BACILLI  319 

Enzymes. — Dysentery  bacilli  do  not  appear  to  produce  extracellular 
proteolytic  enzymes.  They  do  not  liquefy  gelatin,  blood  serum  or 
fibrin,  and  do  not  coagulate  milk.  Wells  and  Corper1  have  demon- 
strated a  lipase  of  moderate  activity  in  the  autolysates  of  dysentery 
bacilli. 

Toxins. — (a)  Exotoxin. — The  nature  of  the  poison  produced  by 
the  Shiga  bacillus,  the  most  virulent  of  the  dysentery  bacilli,  is  a 
matter  of  debate.  Todd,2  Ludke,3  Doerr,4  and  Kraus  and  Doerr5 
state  that  the  organism  produces  a  soluble  (exo-)  toxin  which 
stimulates  antibody  formation  in  suitable  animals;  the  sera  are 
specifically  antitoxic  and  protect  laboratory  animals  against  several 
times  the  fatal  dose  of  the  toxin.  According  to  Kraus  and  Doerr,6 
this  toxin  acts  somewhat  like  that  of  the  diphtheria  bacillus;  the 
lesions  observed  in  the  large  intestine  are  comparable  to  the  lesions 
of  the  diphtheria  bacillus  on  the  tonsils  and  pharynx.  The  nervous 
lesions  are  somewhat  like  those  of  poliomyelitis.  Intravenous  injec- 
tion of  large  doses  in  rabbits  causes  death  in  from  six  to  eight  hours; 
smaller  doses  cause  paresis,  diarrhea,  which  is  frequently  bloody, 
paralysis  of  the  bladder,  hypothermia  and  death  in  one  to  four  weeks. 
Postmortem  there  is  a  mucohemorrhagic  enteritis,  usually  localized 
in  the  cecum.  It  is  stated  that  the  entire  intestinal  tract  is  involved 
in  dogs,  with  the  duodenum  particularly  affected.  Intraperitoneal 
and  subcutaneous  injections  give  a  much  milder  reaction  with  a  pro- 
longed incubation  period.  The  toxin  is  inactivated  by  acids,  but  its 
potency  may  be  partially  restored  when  the  acid  is  neutralized  with 
alkali.  Conradi7  and  others  find  dead  cultures  almost  as  toxic  as  the 
living  bacilli ;  they  call  attention  to  the  toxic  properties  of  autolysates 
(in  sterile  water)  of  the  Shiga  bacillus,  a  fact  which  was  pointed  out 
by  Gay8  some  time  before.  It  is  probable  that  both  soluble  and 
autolytic  poisons  are  concerned  in  the  toxicity  of  filtrates  of  broth 
cultures  of  the  organism.  The  toxic  substances  may  be  obtained  in 
dry  form  by  saturating  the  broth  (freed  from  bacilli  by  filtration 
through  unglazed  porcelain)  with  ammonium  sulphate,  dialyzing  the 
precipitate  to  remove  the  ammonium  salts,  and  evaporation  of  the 

1  Jour.  Infec.  Dis.,  1912,  xi,  388. 

2  Brit.  Med.  Jour.,  December  5,  1902,  ii;   October  4,  1903,  ii. 

3  Jour.  Path,  and  Bact.,  1905,  x,  328. 

4  Cent.  f.  Bakt.,  Orig.,  1905,  xxxviii,  420,  511. 
s  Ztschr.  f.  Hyg.,  1906,  Iv,  1. 

6  Loc.  cit. 

7  Deutsch.  med.  Wchnschr.,  1903,  xxix,  26. 
sPenna.  Med.  Bull.,  1902. 


320  THE  ALCALIGENES— DYSENTERY— TYPHOID 

dialyzed  solution  to  dry  ness  in  vacua.  The  dried  residue  is  very  toxic 
for  rabbits;  0.002  to  0.005  grams  dissolved  in  a  small  amount  of 
sterile  salt  solution  will  usually  kill  these  animals  when  injected 
intravenously.  Smaller  amounts  gradually  increased  stimulate  anti- 
body formation.1  The  antitoxin,  however,  has  little  curative  value, 
for  the  toxin  appears  to  have  a  greater  affinity  for  the  epithelium  of  the 
intestinal  mucosa  and  central  nervous  system  than  it  has  for  the  anti- 
toxin. The  other  members  of  the  dysentery  group  do  not  produce 
soluble  toxic  substances  in  demonstrable  amounts. 

(b)  Endotoxin. — Neisser  and  Shiga2  have  found  that  autolysates 
of  Shiga  bacilli  produce  a  mucohemorrhagic  enteritis  in  rajbbits. 
Besredka,3  Conradi4  and  others  have  also  extracted  substances  from 
the  organisms  by  grinding  them  with  sand,  by  alternate  freezing  and 
thawing  (method  of  MacFadyen  and  Roland),  or  by  autolysis,  which 
in  small  amounts  will  kill  experimental  animals  when  injected  intra- 
venously, intraperitoneally,  or  subcutaneously.  Administration  by 
mouth  is  without  noteworthy  effect.  The  potency  of  the  endotoxin 
is  not  appreciably  impaired  by  an  exposure  to  70°  C.  for  an  hour;  an 
exposure  to  80°  C.  renders  it  inactive.  Conradi5  has  shown  that 
occasional  strains  of  dysentery  bacilli  (Shiga  type)  produce  small 
amounts  of  soluble  hemotoxin. 

Pathogenesis. — Experimental. — Direct  experimental  evidence  of  the 
etiological  relationship  of  the  dysentery  bacillus  to  bacillary  dysentery 
is  afforded  by  a  few  laboratory  accidents  in  which  the  clinical  disease 
has  followed  the  accidental  ingestion  of  cultures  of  dysentery  bacilli. 
The  most  conclusive  experiment,  however,  is  that  reported  by  Strong 
and  Musgrave.6  A  forty-eight-hour  broth  culture  of  B.  dysenteric 
(Shiga  type)  was  swallowed  by  a  condemned  criminal  after  a  dose  of 
sodium  hydrogen  carbonate  was  given  to  neutralize  the  gastric  acidity. 
The  initial  symptoms  of  a  typical  attack  of  bacillary  dysentery  fol- 
lowed after  an  incubation  period  of  thirty-six  hours.  The  organisms 
were  isolated  from  the  mucopurulent,  bloody  feces*.  Ravant  and 
Dopter7  produced  clinical  dysentery  in  an  ape  by  feeding  it  Shiga 
bacilli. 

Human. — Infection  with  the  Shiga  bacillus  is  somewhat  less  com- 
mon in  the  United  States  than  infection  with  the  Flexner  and  other 

1  Todd,  loc.  cit. ;   Kraus  and  Doerr,  loc.  cit. 

2  Deutsch.  med.  Wchnschr.,  1903,  No.  4.         3  Ann.  Inst.  Past.,  April,  1906,  vol.  xxv. 
4  Loc.  cit.  6  Loc.  cit. 

6  Report  of  the  Surgeon-General,  United  States  Army,  1900. 

7  Quoted  by  Kolle  and  Hetsch,  Die  experimentelle  Bakt.,  II.  Aufl.,  i,  304. 


THE  GROUP  OF  THE  DYSENTERY  BACILLI  321 

types  of  the  dysentery  group,  but  far  more  fatal.  Mixed  infections 
in  which  both  Shiga  and  Flexner  bacilli  are  present  are  occasionally 
seen.1  Among  adults  infection  with  the  Flexner  type  of  organism 
tends  to  be  sporadic  in  distribution  and  less  severe  than  infections 
with  the  Shiga  type  which  more  commonly  assume  epidemic  tendencies. 

The  incubation  period  of  bacillary  dysentery  may  be  as  brief  as 
forty-eight  hours,  or  even  less,  and  as  a  rule  there  are  no  distinctive 
prodromal  symptoms.  The  feces,  at  first  watery,  may  be  very  fre- 
quent, as  many  as  twenty  to  thirty  per  diem,  and  become  muco- 
purulent  with  considerable  amounts  of  fresh  blood  mixed  in  them. 
The  organisms  are  present  in  variable  numbers.  Dysentery  bacilli 
do  not  as  a  rule  appear  to  invade  the  blood  stream,  but  at  least  three 
instances  are  on  record  where  pure  cultures  of  the  Shiga  bacillus  have 
been  isolated  antemortem  from  the  general  circulation;2  occasionally 
pure  cultures  of  dysentery  bacilli  may  be  obtained  from  mesenteric 
lymph  nodes  postmortem. 

Lesions. — The  lesions,  which  are  found  chiefly  in  the  large  intestine, 
vary  with  the  severity  and  duration  of  the  disease.  In  the  early  stages 
of  the  disease  there  is  a  severe  catarrhal  inflammation  of  the  mucous 
membrane  of  the  large  intestine  with  some  necrosis  of  the  epithelium, 
associated  with  hyperemia  of  the  mucosa  of  the  small  intestine  as  well. 
The  mesenteric  glands  are  usually  swollen  and  hyperemic.  Later  the 
inflammation  may  become  very  severe;  a  pseudomembrane  may  form 
in  the  large  intestine  with  extensive  superficial  ulceration  of  the 
mucosa.  The  ulcers  do  not  extend  as  a  rule  to  the  submucosa;  conse- 
quently, perforation  is  rare  in  uncomplicated  cases.  The  submucosa, 
however,  may  be  swollen  and  somewhat  edematous. 

The  nervous  symptoms  which  are  a  feature  of  severe  dysentery 
infections  would  suggest  that  in  addition  to  the  intestinal  lesions  there 
may  be  involvement  of  the  nervous  system.  Southard,  McGaffin 
and  Richards3  have  shown  that  in  addition  to  lesions  of  the  intes- 
tinal tract,  the  Shiga  toxin  has  a  special  affinity  for  the  anterior  horn 
ganglion  cells,  thus  explaining  on  a  definite  anatomical  basis  the  ner- 
vous symptoms  which  are  a  feature  of  fatal  cases  of  bacillary  dysentery. 
Dopter4  has  expressed  the  same  opinion.  He  believes  the  toxin  of  the 

1  Kendall,  Bagg,  Day  and  Walker,  loc.  cit. 

2  Rosenthal,  Deutsch.  med.  Wchnschr.,  1903,  No.  6;     Kendall,  Bagg  and  Day,  Boston 
Med.  and  Surg.  Jour.,  1913,  clxix,  741;     Darling  and  Bates,  Am.  Jour.  Med.  Sc.,  1912, 
clxiii,  No.  1. 

3  Boston  Med.  and  Surg.  Jour.,  1909,  clxi,  65,  108. 

4  Loc.  cit.,  p.  77. 

21 


322  THE  ALCALIGENES— DYSENTERY— TYPHOID 

Shiga  bacillus  has  an  elective  affinity  for  the  intestinal  mucosa  of 
the  large  intestine,  and  it  is  the  toxin  secreted  by  the  dysentery  bacilli 
during  their  multiplication  in  the  intestinal  mucous  membrane  which 
induces  the  anatomical  and  nervous  lesions  characteristic  of  the 
disease. 

Animals. — Typical  bacillarv  dysentery  has  not  been  produced  in 
laboratory  animals  by  feeding  the  organisms.  The  intravenous  inocu- 
lation or  intraperitoneal  injection  of  living  or  killed  broth  cultures  of 
Shiga  or  Flexner  bacilli,  however,  are  usually  fatal,  particularly  to 
rabbits.  Vaillard  and  Dopter,1  and  Flexner2  have  shown  that  small 
amounts  of  forty-eight-hour  broth  cultures  of  Shiga  bacilli  introduced 
intravenously  into  young  rabbits  frequently  lead  to  diarrhea,  which 
at  first  is  mucous  in  character;  later  it  becomes  mucosanguineous. 
After  two  or  three  days  symptoms  of  paraplegia  develop.  At  autopsy 
the  large  intestine  is  swollen  and  frequently  edematous.  The  mesen- 
tery is  hyperemic  with  enlarged  glands.  The  intestinal  contents  are 
mucosanguineous  in  character  and  the  intestinal  wall  is  considerably 
thickened.  If  the  animal  survives  for  several  days,  more  advanced 
lesions  are  sometimes  seen,  particularly  beginning  ulcer ation  and 
necrosis.  Flexner  states  that  the  intestinal  lesions  of  bacillary 
dysentery  in  man  and  in  animals  are  probably  due,  in  part  at  least, 
to  the  direct  action  of  the  dysentery  toxin. 

Immunity  and  Immunization. — Shiga3  and  others  have  succeeded  in 
immunizing  laboratory  animals,  particularly  rabbits,  guinea-pigs,  and 
horses,  with  dysentery  bacilli,  beginning  by  injecting  killed  cultures 
of  these  organisms,  first  with  very  small  amounts  which  are  slowly 
and  cautiously  increased,  finally  with  living  bacilli.  It  is  difficult  to 
immunize  animals  because  of  the  toxicity  of  the  organism.  The  sera 
of  these  animals  contain  specific  agglutinins,  lysins,  precipitins,  and 
opsonins,  frequently  of  high  potency.  According  to  Todd,  and  Kraus 
and  Doerr,4  specific  antitoxins  are  also  demonstrable  in  the  sera  of 
these  animals,  particularly  in  animals  immunized  to  the  Shiga  bacillus. 
The  agglutinins  which  are  specific  for  the  type  of  organism  used  in 
immunization  are,  according  to  Dopter,5  as  a  rule  of  greater  potency 
when  killed  cultures  exclusively  are  used  for  immunizing.  In  .thor- 
oughly immunized  animals  the  agglutinins  may  be  active  even  in 
dilutions  of  1  to  5000. 

1  Ann.  Inst.  Past.,  1903,  p.  472. 

2  Jour.  Exp.  Med.,  1906,  vol.  viii. 

3  Ztschr.  f.  Hyg.,  1902,  xli,  355. 

4  Loc.  cit.  5  Loc.  cit.,  p.  84, 


THE  GROUP  OF  THE  DYSENTERY  BACILLI  323 

Specific  bacteriolysins  have  been  demonstrated  in  immune  sera 
in  vitro  by  Shiga1  and  in  vivo  by  Kruse.2  Specific  precipitins,  which 
in  dilutions  of  1  to  10  or  greater  will  produce  a  precipitate  in  broth 
filtrates  of  the  homologous  strain,  but  not,  as  a  rule,  for  other  types 
of  the  dysentery  bacilli  are  also  found.  The  sera  of  patients  who 
have  recovered  from  attacks  of  bacillary  dysentery  usually  contain 
specific  agglutinins  which  are  active  even  in  dilutions  of  1  to  50. 
Specific  precipitins,  lysins,  and  opsonins  are  also  demonstrable  in  the 
sera  of  these  patients. 

Therapy. — Attempts  to  immunize  man  with  vaccines,  both  mono- 
and  polyvalent,3  sensitized  vaccines  (bacteria  which  have  been  in 
contact  with  antidysentery  serum,  then  centrifugalized,  washed,  and 
suspended  in  salt  solution,  according  to  the  method  of  Besredka  and 
of  Gay),  and  the  use  of  antisera,  usually  derived  from  immunized 
horses,  have  not  been  generally  successful,  although  a  few  favorable 
results  have  been  recorded. 

Bacteriological  Diagnosis. — (a)  Agglutinin  Reaction. — The  sera  of 
normal  individuals  rarely  agglutinate  dysentery  bacilli  in  dilutions 
greater  than  1  to  10,  although  Dopter4  states  that  the  Flexner  organ- 
ism may  be  clumped  with  the  serum  of  apparently  normal  individuals 
in  a  dilution  greater  than  1  to  10.  For  this  reason  agglutination  tests 
should  be  made  in  a  dilution  of  1  to  20  to  1  to  30  with  the  Shiga 
organism,  and  1  to  80  to  1  to  100  with  the  Flexner  strain  in  each 
case  examined,  since  one  or  the  other  organism,  or  both,  may  be 
present  in  typical  cases  of  bacillary  dysentery.  Agglutinins  do  not 
as  a  rule  appear  in  mild  cases,  and  in  severe  cases  they  are  not 
demonstrable  until  from  the  seventh  to  the  tenth  day  on  the  average. 

The  serum  of  dysentery  carriers,  both  those  giving  a  history  of  a 
previous  attack  and  those  with  the  negative  dysentery  history,  fre- 
quently agglutinates  either  with  Shiga  or  Flexner  bacilli.  The  agglu- 
tination reaction,  therefore,  is  not  conclusive  for  clinical  diagnosis 
unless  a  negative  reaction  is  obtained  early  in  the  disease  followed 
by  a  positive  reaction  on  or  after  the  seventh  to  the  tenth  day. 

(6)  Isolation  of  Dysentery  Bacilli  from  the  Feces. — Dysentery  bacilli 
do  not  invade  the  blood  stream  as  a  rule,  and  they  are  not  found 
in  the  urine.  The  bacteriological  diagnosis,  therefore,  depends  upon 

1  Loc.  cit. 

2  Deutsch.  med.  Wchnschr.,  1902. 

3  Shiga,  Deutsch.  med.  Wchnschr.,  1901,  Nos.  43  and  45;     Kruse,  ibid.,  1903,  Nos.  1 
and    3. 

4  Loc.  cit.,  p.  91. 


324  THE  ALCALIGENES— DYSENTERY— TYPHOID 

the  isolation  of  the  organisms  from  the  feces  and  their  identification 
by  cultural  and  serological  reactions. 

A  bit  of  blood-stained  mucus  offers  the  best  material  for  isolation 
of  the  organisms:  it  should  be  washed  two  or  three  times  in  sterile 
salt  solution  to  remove  extraneous  organisms  as  far  as  possible,  for 
experience  has  shown  that  dysentery  bacilli  are  frequently  enclosed 
in  mucus.  The  mucus  is  then  macerated  in  sterile  broth,  and  if 
possible  incubated  for  one  or  two  hours  at  37°  C.  It  is  then  spread 
upon  the  surface  of  Endo-plates  and  incubated  for  eighteen  to  twenty- 
four  hours  at  37°  C.  The  colonies  are  precisely  similar  to  those  of 
typhoid  and  paratyphoid  bacilli;  the  final  identification  of  the  dysen- 
tery bacilli  is  made  by  their  cultural  reactions  (see  page  316)  and  by 
agglutination  with  specific  sera  of  high  potency.  The  rapid  method 
of  isolating  and  identifying  typhoid  bacilli  described  on  page  338  is 
equally  applicable  to  dysentery  bacilli.  The  possibility  of  carriers 
should  be  borne  in  mind  when  mild  and  atypical  cases  are  under 
consideration . 

Dissemination  and  Prophylaxis. — Dysentery  bacilli  appear  to  be 
widely  distributed  in  certain  areas  of  the  temperate  zone,  and  out- 
breaks occur  at  varying  intervals.  Interepidemic  years  are  occa- 
sionally characterized  by  considerable  numbers  of  atypical,  mild 
cases,  and  carriers  are  not  uncommon.1 

The  organisms  enter  the  body  through  the  mouth  and  intestinal 
tract,  and  leave  it  in  the  feces;  consequently  the  method  of  trans- 
mission of  the  disease  is  similar  to  that  of  typhoid  and  other  excre- 
mentitious  disorders.  There  is  some  evidence  that  the  disease  may 
be  milk-borne;  exclusively  breast-fed  infants  are  rarely  or  never 
infected;  bottle-fed  babies  of  the  same  age  may  be  infected  in  relatively 
large  numbers  during  years  which  exhibit  an  epidemic  tendency  of 
bacillary  dysentery.  Zinsser2  has  produced  evidence  in  favor  of  the 
occasional  milk  transmission.  The  organism  may  also  reach  the  body 
by  direct  transmission  through  carriers,  in  hospitals,  and  through 
contaminated  water  and  food.  Flies  may  also  play  a  part  in  the  spread 
of  the  disease. 

The  precautions  to  be  observed  are  those  for  any  intestinal  infec- 
tion. 

1  Kendall,  Boston  Med.  and  Surg.  Jour.,  1913,  clxix,  7493;   ibid.,  May  20,  1915. 

2  Proc.  New  York  Path.  Soc.,  1907. 


TYPHOID  BACILLUS  325 


TYPHOID    BACILLUS. 


Historical. — Typhoid  bacilli  were  first  seen  in  sections  of  tissue 
from  autopsies  by  Klebs  in  1876.  Somewhat  later  Eberth1  success- 
fully demonstrated  them  in  sections  of  mesenteric  glands,  lymph 
nodes  and  the  spleen  by  the  use  of  the  recently  introduced  tissue 
stains.  Gaffky2  first  isolated  the  organisms  in  pure  culture  and 
established  their  probable  etiological  relationship  to  typhoid  fever. 
Later  investigations  with  more  refined  methods  have  completely 
substantiated  Gaffky's  observations. 

Morphology — Typhoid  bacilli  are  rod-shaped  organisms  of  moderate 
size,  measuring  from  0.5  to  0.8  microns  in  diameter  and  from  1 
to  3  microns  in  length.  The  dimensions  vary  within  the  limits 
given  upon  different  media,  the  organisms  being  as  a  rule  somewhat 
longer  in  fluid  media  than  upon  solid  media.  Elongated  rods  and  even 
filaments  are  occasionally  found  in  old  gelatin  and  potato  cultures. 
The  bacilli  have  rounded  ends  and  occur  as  a  rule  singly  or  in  pairs. 
They  are  actively  motile,  particularly  in  young  cultures  grown  in 
0.1  per  cent,  dextrose  broth;  plain  broth  cultures  are  usually  more 
sluggish.  Each  organism  possesses  characteristically  from  eight  to 
ten  peritrichic  flagella;  rarely  as  many  as  twenty  may  be  attached  to 
a  single  organism.  The  flagella  are  somewhat  wavy  in  outline  and 
measure  from  6  to  8  microns  in  length.  No  spores  are  produced 
It  was  formerly  held  that  typhoid  bacilli  formed  no  capsules.  Car- 
pano,3  and  Gay  and  Claypole,4  however,  have  demonstrated  capsules 
around  typhoid  bacilli  grown  in  blood  media. 

The  organisms  stain  readily  with  ordinary  anilin  dyes  and  they 
are  Gram-negative. 

Isolation  and  Culture. — The  typhoid  bacillus  grows  readily  upon  the 
ordinary  media.  Colonies  on  agar  plates  are  round,  colorless,  flat,  and 
nearly  transparent;  they  attain  a  diameter  of  from  0.5  to  1.5  mm. 
after  eighteen  to  twenty-four  hours'  incubation  at  37°  C.  Devel- 
opment in  gelatin  is  less  rapid,  and  the  colonies  after  two  to  three 
days'  incubation  at  20°  C.  are  somewhat  brownish  in  color.  A  uniform 
turbidity  is  produced  in  plain  broth  after  eighteen  hours'  growth  at 
37°  C.;  development  in  dextrose  broth  is  more  intense,  but  after  five 
to  seven  days  it  ceases  and  the  organisms  die,  due  to  the  accumula- 

1  Virchows  Arch.,  1880,  Ixxxi,  58;  1881,  Ixxxiii,  486. 

2  Mitt.  a.  d.  kais.  Gesamte,  1884,  ii,  370. 

3  Cent.  f.  Bakt.,  Orig.,  1913,  Ixx,  42. 

4  Arch.  Int.  Med.,  1913,  xii,  624. 


326  THE  ALCAL1GENES— DYSENTERY— TYPHOID 

tion  of  acid.  Growth  is  luxuriant  in  milk,  but  there  is  little  chemical 
change  in  the  composition  of  the  medium  as  the  result  of  the  growth.1 
Two  types  of  reaction  are  observed  in  litmus  milk:  (a)  The  reaction 
becomes  slightly  acid,  turning  the  litmus  to  a  lilac  color  which  per- 
sists. This  is  much  more  common  than  (6) ;  the  milk  becomes  slightly 
acid,  as  in  "a,"  then  it  becomes  slowly  but  progressively  alkaline. 
Relatively  few  authentic  strains  of  typhoid  bacilli  appear  to  produce 
the  transient  acidity  in  this  medium.  At  one  time  potato  was  regarded 
as  an  important  differential  medium  for  the  recognition  of  the  typhoid 
bacillus.  The  "invisible  growth"  described  by  Gaffky2  is  now  known 
to  be  dependent  largely  upon  the  reaction;  potatoes  having  an  acid 
reaction  give  this  invisible  growth;  old  potatoes  which  usually  have 


FIG.  46. — Bacillus  typhosus,  flagella  stain. 

a  slightly  alkaline  reaction  give  a  heavy,  brownish  growth  much  like 
that  of  the  colon  bacillus.  The  addition  of  small  amounts  of  alkali, 
as  sodium  carbonate,  to  potato  prior  to  inoculation  makes  the  growth 
visible  and  brown;  the  addition  of  a  small  amount  of  organic  acid  to 
the  medium  usually  results  in  the  development  of  the  invisible  type 
of  growth. 

The  typhoid  bacillus  is  an  aerobic,  facultatively  anaerobic  organism, 
whose  minimal  temperature  of  growth  is  about  8°  C.;  development  is 
maximal  at  37°  C.,  and  ceases  when  the  culture  is  exposed  to  tem- 
peratures above  43°  to  44°  C.  An  exposure  of  ten  to  twenty  minutes 
at  60°  C.  will  kill  the  naked  organisms;  a  longer  exposure  at  a  higher 
temperature  is  required  to  kill  them  when  they  are  suspended  in  organic 

1  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Assn.,  1914,  xxxvi,  1958, 

2  Loc.  cit. 


TYPHOID  BACILLUS  327 

matter,  as  feces.  Cultures  exposed  to  temperatures  from  0°  C.  to 
—  10°  C.  for  three  months  occasionally  contain  viable  organisms. 
Alternate  freezing  and  thawing  is  more  fatal  than  simple  freezing. 
The  typhoid .  bacillus  dies  out  rather  rapidly  in  potable  water,  less 
rapidly  in  sterilized  potable  water.  The  addition  of  organic  matter, 
particularly  of  fecal  origin,  appears  to  promote  longevity  somewhat. 
The  observations  of  Jordan,  Russell  and  Zeit1  would  indicate  that  a 
large  percentage  of  organisms  exposed  in  potable  water  die  within 
three  days.  Kersten2  has  shown  that  typhoid  bacilli  will  develop 
with  considerable  rapidity  in  raw  milk.  The  bacilli  may  remain  alive 
in  soil  for  several  months,  provided  they  are  shielded  from  direct 
sunlight,  and  they  may  resist  drying  under  similar  conditions  for 


FIG.  47. — Bacillus  typhosus,  bouillon  culture.      X  1000. 

several  weeks.  A  maximum  exposure  of  from  four  to  eight  hours  to 
direct  sunlight  in  the  months  of  June,  July  and  August  (Northern 
Hemisphere)  usually  kills  the  organisms.  Mercuric  chloride  1  to  1000 
kills  the  naked  germs  in  about  ten  minutes;  5  per  cent,  carbolic  acid 
kills  them  in  from  five  to  ten  minutes,  as  a  rule. 

Products  of  Growth. — The  typhoid  bacillus  liberates  ammonia  from 
protein  in  sugar-free  media,  and  forms  small  amounts  of  non-volatile 
alkaline  products  as  well.  The  reaction,  therefore,  becomes  progres- 
sively alkaline.  A  radical  change  in  the  nature  of  the  products  of 
metabolism  occurs  when  the  bacilli  are  grown  in  protein  media  con- 
taining utilizable  carbohydrates,  as  dextrose  or  mannite.  The  reaction 
becomes  strongly  acid,  due  to  the  fermentation  of  the  sugar.  The 

1  Jour.  Infec.  Dis.,  1904,  i,  641. 

2  Arb.  a.  d.  kais.  Gesarat.,  1909,  xxx,  341. 


328  THE  ALCALIGENES— DYSENTERY— TYPHOID 

protein  under  these  conditions  is  left  unattacked  except  for  minute 
amounts  necessary  to  supply  the  nitrogenous  requirements  of  the 
organism.  The  acids  formed  are  chiefly  lactic  acid,  together  with 
smaller  amounts  of  formic  acid.1  Indol  or  phenols  are  not  formed 
in  ordinary  media,  but  Peckham2  has  shown  that  indol  may  be  pro- 
duced in  protein  media  of  special  composition. 

The  essential  cultural  characters  of  B.  typhosus  are  indicated  in 
the  table  on  page  316.  Culturally  Bacillus  typhosus.  is  relatively 
inert;  it  does  not  produce  proteolytic  enzymes  which  liquefy  gelatin, 
blood  serum  or  fibrin.  A  fat-splitting  ferment  has  been  demonstrated 
in  autolyzed  typhoid  bacilli  by  Wells  and  Corper.3  An  esterase 
which  liberates  butyric  acid  from  ethyl  butyrate  is  detectable  in  sterile 
filtrates  of  plain  and  dextrose  broth  cultures  of  the  organism.4 

Typhohemolysin  (typholysin) .  Castellani,5  and  E.  Levy  and  P. 
Levy6  have  found  that  filtrates  of  (sugar-free)  broth  cultures  of  typhoid 
bacilli  are  hemolytic.  They  appear  to  have  demonstrated  specific 
antihemolytic  properties  in  the  blood  of  animals  injected  with  hemo- 
lytic filtrates,  thus  meeting  the  objection  that  the  hemolysis  might  be 
due  to  the  alkalinity  of  the  medium  itself.  There  is  no  evidence  at 
present  which  would  suggest  that  this  hemolysin  plays  any  impor- 
tant part  in  typhoid  infections  of  man.  The  typholysin  is  relatively 
thermostabile. 

Toxins. — A  soluble  toxin  has  never  been  satisfactorily  demon- 
strated among  the  products  of  growth  of  the  typhoid  bacillus,  and 
the  consensus  of  opinion  at  the  present  time  is  in  favor  of  the  view 
that  the  principal  toxic  substance  of  the  organism  is  an  endotoxin. 
The  endotoxin  has  been  studied  with  special  thoroughness  by  Mac- 
Fadyen  and  Roland,7  and  Besredka.8  It  has  been  obtained  in  various 
ways:  by  grinding  the  organisms  with  sand,  by  freezing  in  liquid  air 
and  triturating,  or  by  autolysis  of  the  bacilli  in  sterile  distilled  water. 
Relatively  small  amounts  of  endotoxin  obtained  by  any  of  these 
methods  will  usually  kill  guinea-pigs.  No  antitoxin  has  been  produced 
in  the  sera  of  animals  inoculated  with  gradually  increasing  amounts 
of  this  endotoxin. 


1  Kendall,  Jour.  Med.  Research,  1911,  xxiv,  411;    1912,  xxv,  117.     Boston  Med.  and 
Surg.  Jour.,  1911,  Ixiv,  288.     Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Assn.,  1913, 
xxxv,  1214. 

2  Jour.  Exper.  Med.,  1897,  ii,  549.  3  Jour.  Infec.  Dis.,  1912,  xi,  388. 
4  Kendall  and  Simonds,  Jour.  Infec.  Dis.,  1914,  xv,  354. 

6  Lancet,  February  15,  1902.  •  Cent.  f.  Bakt.,  1901,  xxx,  405. 

7  Cent.  f.  Bakt.,  Orig.,  1903,  xxxiv,  618,  765;  MacFadyen,  ibid.,  1903,  xxxv,  415. 

8  Ann.  Inst.  Past.,  1905-1906. 


TYPHOID  BACILLUS  329 

Typhoid  Fever. — Pathogenesis. — Experimental — Typhoid  fever  is  a 
disease  of  man  only,  and  until  recently  rigorous  experimental  proof 
that  the  typhoid  bacillus  is  the  specific  cause  of  this  infection  has  been 
lacking.  The  evidence  of  the  etiological  relationship  of  the  typhoid 
bacillus  is  of  two  kinds:  (1)  a  few  cases  where  laboratory  attendants 
have  accidentally  or  purposely  swallowed  cultures  of  typhoid  fever 
and  have  developed  the  disease ;  (2)  experiments  of  Metchnikoff  and 
Besredka.1 

The  experiments  of  Metchnikoff  and  Besredka  appear  to  be  con- 
clusive. They  produced  typhoid  fever  in  anthropoid  apes  by  feeding 
the  animal  food  infected  with  fecal  material  containing  typhoid  bacilli. 
The  animals  (fifteen  in  all)  developed  fever  and  diarrhea  after  eight 
days,  and  typhoid  bacilli  were  isolated  from  the  blood  stream  on  the 
tenth  day.  Three  died.  Specific  agglutinins  were  demonstrable  in 
the  blood  serum,  and  the  clinical  picture  was  essentially  that  of  typical 
typhoid  fever.  These  observers  ruled  out  the  possibility  of  a  filterable 
virus. 

Pathogenesis  in  Man. — Portal  of  Entry. — Typhoid  bacilli  enter  the 
body  through  the  mouth  and  pass  through  the  gastro-intestinal  tract. 
They  lodge  in  lymphatic  tissue  of  the  intestines,  particularly  Peyer's 
patches,  then  invade  the  general  lymphatic  system  and  spleen,  and 
are  found  in  the  blood,  especially  during  the  first  week  of  the  clinical 
disease.  Typhoid  fever,  therefore,  is  a  bacteremia.  Rose  spots,  which 
are  frequently  found  on  the  abdomen  during  the  first  week  of  the 
clinical  disease,  contain  colonies  of  typhoid  bacilli  which  are  localized 
in  the  subcutaneous  tissue.2  Characteristic  lesions  are  found  in  Peyer's 
patches  which  at  first  are  swollen  and  hyperemic.  After  a  few  days 
the  glands  become  rather  pale,  caused,  in  part  at  least,  by  hyperplasia 
of  the  lymphoid  and  endothelioid  cells,  which  cuts  off  the  blood  supply 
in  whole  or  in  part,  leaving  these  areas  even  more  prominent  (medul- 
lary swelling).3  Necrosis  then  commences  and  the  glands  gradually 
become  yellowish  in  color  and  'softer  in  consistency.  Soon  the  necrosis 
ceases  rather  abruptly  as  immunity  checks  the  process  and  the  necrotic 
tissue  then  sloughs  away,  leaving  a  somewhat  irregular  elongated  ulcer 
which  usually  extends  to  or  through  the  muscular  layer  of  the  intestine. 
About  the  end  of  the  third  week  scar  tissue  begins  to  appear  in  these 
ulcers,  which  in  time  practically  fills  up  the  original  area,  leaving  the 

1  Ann.  Inst.  Past.,  March  25,  1911;  xxv,  193,  865. 

2  Richardson,  Philadelphia  Med.  Jour.,  March,  1900.  (Special  Typhoid  Fever  Number.) 
8  Mallory,  Jour.  Exp.  Med.,  1898,  iii,  No.  6,  p.  611. 


330  THE  ALCALIGENES— DYSENTERY— TYPHOID 

site  of  the  ulcer  marked  by  a  somewhat  depressed  cicatrix.  Occasion- 
ally secondary  infection  of  the  ulcers  results  in  perforation  or  hemor- 
rhage, and  sometimes  an  uninfected  ulcer  may  erode  through  a 
blood  vessel,  causing  hemorrhage.  It  should  be  remembered  that 
typhoid  ulcers  tend  to  run  along  the  long  axis  of  the  intestine,  whereas 
tuberculous  ulcers,  on  the  contrary,  run  transversely,  following  the 
course  of  the  lymphatics. 

In  addition  to  the  intestinal  lesions,  there  is  in  typhoid  fever  an 
acute  splenic  tumor  with  a  great  proliferation  of  typhoid  bacilli  in 
this  organ.  Foci  of  typhoid  bacilli  are  commonly  found  also  in  the 
kidneys  and  the  liver,  mesenteric  lymph  nodes,  less  commonlv  in 
lungs,  meninges,  bone  marrow,  certain  muscles  and  the  tonsils.  Paren- 
chymatous  degeneration  of  the  heart,  liver  and  kidneys  is  common, 
as  is  a  catarrhal  inflammation  of  the  respiratory  tract  and  a  severe 
inflammation  of  the  entire  intestinal  mucous  membrane.  Somewhat 
uncommonly,  typhoid  cases  have  been  recorded  in  which  there  are  no 
intestinal  lesions.  In  these  cases  it  would  appear  that  the  disease  is 
septicemic  in  character.1  In  typhoid  fever  there  is  leucopenia,  due 
apparently  to  some  interference  with  the  activity  of  the  bone  marrow. 
The  febrile  reaction  is  usually  attributed  to  the  liberation  of  endotoxin 
from  typhoid  bacilli,  which  are  dissolved  in  the  blood  stream  by 
specific  lysins.  This  toxin  exhibits  both  a  general  and  local  reaction. 
The  general  reaction  is  characterized  chiefly  by  fever  and  symptoms 
of  generalized  toxemia;  the  local  reaction  is  particularly  marked  in 
those  areas  where  typhoid  bacilli  undergo  solution,  as  in  the  spleen 
and  Peyer's  patches. 

Various  complications  of  typhoid  fever  are  occasionally  reported, 
caused  by  the  localization  of  typhoid  bacilli  either  alone  or  in 
association  with  other  bacteria,  as  the  streptococcus,  staphylococcus, 
or  pneumococcus,  in  various  organs.  Peritonitis,  usually  following 
perforation  of  an  ulcer  in  the  intestinal  wall,  is  one  of  the  most  severe 
of  these  complications.  Abscess  formation  in  various  deep-seated 
organs,  as  the  spleen  and  psoas  muscle,  is  not  uncommon.  Broncho- 
pneumonia,  pleurisy,  pericarditis,  osteitis,  and  inflammation  of  the 
membranes  of  the  cord  (meningitis)  and  brain  have  also  been  attributed 
to  the  typhoid  bacillus. 

Carriers. — Typhoid  bacilli  can  not  be  isolated  from  the  majority 
of  typhoid  patients  after  the  fifth  week  of  the  disease.  In  a  small 

1  Possett,  Atypische  Typhusinfektion.  Lubarsch  and  Ostertag,  Ergebn.  d.  allgem. 
Pathol.,  1912,  xvi,  184. 


TYPHOID  BACILLUS  331 

percentage  of  cases,  however,  the  organisms  may  be  excreted  in  the 
urine,  or  more  commonly  in  the  feces,  for  months  or  even  years  after 
recovery.  Thus,  Philipowicz1  isolated  typhoid  bacilli  from  a  case 
of  cholecystitis  who  had  had  typhoid  fever  thirty-eight  years  previous 
to  the  operation.  In  this  case  very  few  typhoid  bacilli  were  present 
in  the  feces,  and  it  is  probable  that  the  few  organisms  were  over- 
whelmed by  the  intestinal  bacteria  during  their  passage  through  the 
intestinal  tract.  From  1  to  4  per  cent,  of  all  typhoid  cases  which 
recover  appear  to  become  fecal  typhoid  carriers;  a  smaller  percentage 
become  urinary  carriers.  No  history  of  typhoid  fever  can  be  elicited 
from  some  of  these  carriers,  and  the  supposition  is  that  either  the 
carrier  had  in  the  past  a  mild  unrecognized  case,  or  less  commonly 
that  the  organism  had  become  acclimatized  in  the  intestinal  tract 
without  inducing  disease.  Many  carriers  give  a  positive  Widal  reaction. 

The  residual  focus  of  typhoid  bacilli  in  carriers  is  usually  the  gall- 
bladder and  the  ducts  of  the  gall-bladder,  less  commonly  the  urinary 
bladder.  From  the  gall-bladder  the  organisms  pass  in  irregular  num- 
bers into  the  intestinal  tract;  occasionally  in  sufficient  numbers  to  be 
demonstrable  in  the  feces.  A  considerable  proportion  of  operations 
for  cholecystitis  and  gall-stones — the  greater  majority  being  among 
women — give  positive  typhoid  cultures  when  the  contents  are  examined 
bacteriologically. 

Pathogenesis  in  Animals. — All  animals,  except  possibly  anthropoid 
apes,  are  naturally  immune  to  typhoid  fever,  and  inoculation  of  old 
laboratory  cultures  of  typhoid  bacilli  into  laboratory  animals  is 
usually  without  noteworthy  effect;  virulent  cultures  of  typhoid  bacilli, 
particularly  those  produced  by  repeated  passage  through  laboratory 
animals,  may  produce  peritonitis  and  death  when  they  are  introduced 
into  the  animals  by  the  intraperitoneal  route.  The  infection,  how- 
ever, does  not  resemble  typhoid  fever.  The  lesions  observed  post- 
mortem are  marked  congestion  of  the  abdominal  organs,  particularly 
the  spleen,  kidneys  and  liver,  as  well  as  involvement  of  the  intestinal 
lymph  apparatus;  the  thoracic  organs  are  less  involved  as  a  rule. 

The  organisms  may  be  recovered  from  the  peritoneal  fluid,  the  blood 
stream,  and  from  various  abdominal  organs.  Gay  and  Claypole2 
have  succeeded  in  inducing  with  great  regularity  the  carrier  state  in 
rabbits  by  injecting  into  them  typhoid  bacilli  which  have  been  grown 
for  several  successive  transfers  on  agar  overlaid  with  fresh  defibrinated 

1Wien.  klin.  Wchnschr.,  1911,  1802. 
2  Arch.  Int.  Med.,  December,  1913. 


332  THE  ALCALIGENES— DYSENTERY— TYPHOID 

rabbit's  blood.  They  found  that  the  typhoid  bacilli  localize  them- 
selves in  the  gall-bladders  of  the  rabbits,  and  that  they  may  from  time 
to  time  invade  the  blood  stream.  In  a  more  recent  communication1 
they  have  shown  that  the  carrier  state  occurs  much  less  frequently 
if  the  animals  are  immunized  with  their  dried  sensitized  vaccine. 

Antibody  Production. — Animals  may  be  immunized  by  repeated 
injections  of  typhoid  bacilli  to  such  a  degree  that  they  will  successfully 
resist  several  times  the  original  fatal  dose  of  these  organisms.2  Suc- 
cessive injections  of  typhoid  bacilli  stimulate  antibody  formation 
in  horses,  rabbits,  guinea-pigs,  and  other  animals.  Of  these  anti- 
bodies, the  lysins  and  agglutinins  may  be  produced  in  high  potency 
if  the  injections  are  continued  long  enough.  Other  antibodies,  opsonins 
and  precipitins  particularly,  are  also  produced.  Gay  and  Claypole3 
have  produced  experimental  evidence  indicating  that  the  titre  of  the 
specific  agglutinins  which  develop  during  the  process  of  immunization 
of  rabbits  affords  no  indication  of  the  degree  of  protection  attained  by 
the  immunizing  process. 

Protective  Immunization. — As  a  rule,  one  attack  of  typhoid  fever 
confers  immunity;  subsequent  attacks  are  unusual. 

During  the  last  few  years  definite  progress  has  been  made  in  the 
protective  immunization  of  human  beings,  both  by  the  use  of  killed 
cultures  of  typhoid  bacilli  and  by  live  cultures.  The  vaccine  treat- 
ment for  typhoid  fever  is  the  best  known  and  the  most  widely  prac- 
ticed. The  procedure  is  to  grow  typhoid  bacilli  on  agar  slants,  wash 
them  off  with  sterile  physiological  salt  solution,  kill  them  by  heating 
to  60°  C.  for  an  hour,  standardizing  the  suspension  of  typhoid  bacilli, 
and  injecting  as  a  first  dose  five  hundred  million  killed  typhoid  organ- 
isms. After  an  interval  of  seven  to  ten  days  a  second  injection  of  a 
billion  killed  typhoid  bacilli  is  made,  and  after  an  equal  interval  a 
third  and  last  injection  of  a  billion  killed  typhoid  bacilli  is  made. 
In  about  20  per  cent,  of  the  cases  injected  general  symptoms  which 
consist  of  a  febrile  reaction  and  malaise  develop,  accompanied  by 
local  symptoms  of  pain,  redness,  and  swelling  at  the  site  of  inoculation. 
These  symptoms  may  appear  after  the  second  or  even  after  the  third 
injection.  It  is  customary  to  make  the  inoculation  about  four  o'clock 
in  the  afternoon,  so  that  the  patient  in  the  majority  of  cases  sleeps 
through  the  general  symptoms. 

1  Arch.  Int.  Med.,  1914,  xiv,  671. 

2  See  Gay  and  Claypole,  Arch.  Int.  Med.,  1914,  xiv,  671,  for  essential  details. 

3  Loc.  cit. 


TYPHOID  BACILLUS  333 

The  immunity  produced  is  generally  considered  to  be  relatively 
complete  for  from  six  months  to  a  year.  It  must  be  remembered  that 
for  at  least  three  weeks  following  the  vaccination  there  is  a  diminution 
in  the  resistance  of  the  individual  to  typhoid  fever;  consequently, 
typhoid  vaccination  should  not  be  undertaken  if  there  is  a  possibility 
of  exposure  to  typhoid  during  this  period.  Vaccination  is  also  very 
undesirable  if  it  is  performed  during  the  incubation  period  of  typhoid 
fever.  It  should  be  practiced  only  on  perfectly  healthy  subjects  free 
from  all  general  and  local  organic  defects  or  infections,  particularly 
tuberculosis.  Nurses,  ward  orderlies,  doctors,  and  those  engaged  in 
the  care  of  typhoid  patients  are  particularly  likely  to  benefit  by  these 
inoculations.  Gay  and  Claypole1  have  demonstrated  experimentally 
that  a  satisfactory  degree  of  protection  may  be  attained  in  animals 
by  three  injections,  at  intervals  of  two  days  each,  of  a  dried  sensitized 
vaccine.  Observations  upon  man  immunized  with  this  vaccine 
indicate  that  the  reactions  are  milder  and  the  whole  process  can  be 
completed  within  a  week,  thus  diminishing  very  materially  the  time 
element  which  has  been  an  important  factor  in  the  past.  It  is  very 
probable  that  the  period  of  increased  susceptibility  to  infection  may 
be  decidedly  shortened  as  well. 

Vaccination  with  Living  Cultures. — Metchnikoff  and  Besredka2 
found  that  the  subcutaneous  injection  of  living  sensitized  cultures 
produced  an  immunity  in  anthropoid  apes  which  was  apparently 
as  definite  as  that  produced  by  an  actual  attack  of  typhoid  fever. 
The  organisms  were  shown  not  to  appear  in  the  urine  or  feces  or  blood 
when  introduced  subcutaneously.  They  were  unable  to  induce 
immunity  in  the  chimpanzee  with  killed  cultures  of  typhoid  bacilli 
or  with  autolysates  of  killed  cultures.  Having  in  mind  the  efficiency 
of  living  cultures,  they3  attempted  the  vaccination  of  man  with  living 
cultures  of  the  typhoid  bacillus.  They  used  sensitized  cultures  which 
appeared  to  cause  only  a  feeble  local  reaction  and  no  general  reaction 
in  the  chimpanzee,  in  preference  to  non-sensitized  living  cultures, 
which  they  found  produced  rather  intense  local  and  general  reac- 
tions. The  vaccine  was  prepared  by  emulsifying  agar  cultures  of 
typhoid  bacilli  in  normal  salt  solution  and  permitting  the  organisms 
to  remain  in  contact  with  antityphoid  serrm  for  twenty-four  hours 
at  37°  C.  The  organisms  are  then  removed  by  centrifuging,  washed 

1  Loc.  cit. 

2  Ann.  Inst.  Past.,  1913,  xxvii,  597.     Besredka,  Ann.  Inst.  Past.,  1913,  xxvii,  607. 

3  Semaine  Med.,  July  24,  1912,  355. 


334  THE  ALCALIGENES— DYSENTERY— TYPHOID 

repeatedly,  then  re-emulsified  in  normal  saline  solution  and  heated 
to  50°  C.  for  thirty  minutes,  then  standardized  in  the  usual  manner. 
Nearly  eight  hundred  people  have  been  vaccinated  with  these  sen- 
sitized living  cultures;  the^  local  reaction  was  slight  in  each  instance, 
and  only  exceptionally  was  there  any  general  reaction.  A  careful 
examination  of  the  blood,  urine  and  feces  of  sixty-four  of  these  cases 
failed  to  show  typhoid  bacilli,  which  would  suggest  that  individuals 
vaccinated  with  living  typhoid  bacilli  neither  develop  typhoid  fever 
nor  become  carriers.  The  cases  are  too  few  in  number  to  compare 
statistically  with  the  cases  vaccinated  with  killed  cultures.  Gay  and 
Claypole1  have  taken  issue  with  Metchnikoff  upon  this  point  and  their 
experiments  indicate  that  their  sensitized  dried  vaccine  may  be 
equally  or  more  efficient  without  the  theoretical  dangers  which  attend 
the  use  of  living  bacilli. 

Various  attempts  have  been  made  to  induce  passive  immunity  to 
typhoid  infection  by  the  injection  of  sera  obtained  from  horses  which 
have  received  numerous  injections  of  typhoid  bacilli  or  their  soluble 
products.  The  results  have  on  the  whole  not  been  encouraging.  Gay 
and  Force2  have  applied  a  preparation  of  typhoid  bacilli  ("  typhoidin") 
made  like  Koch's  old  tuberculin,  by  the  von  Pirquet  method,  to 
patients  that  have  recovered  from  typhoid  fever  and  to  those  who  have 
been  vaccinated  with  typhoid  bacilli.  They  find  that  95  per  cent,  of 
recovered  cases  from  typhoid  (20  cases  out  of  21  examined)  gave  a 
clear-cut  cutaneous  reaction.  One  case  had  typhoid  forty-one  years 
previously.  The  reaction  was  negative  in  85  per  cent,  of  individuals 
not -giving  a  history  of  typhoid  (and  presumably  not  vaccinated) — 41 
cases  tested.  The  9  cases  (15  per  cent.)  that  gave  a  positive  reaction 
were  suspected  to  have  had  a  mild  undiagnosed  attack.  Several, 
but  not  all,  of  those  vaccinated  within  four  years  (9  out  of  15)  gave 
a  positive  reaction.  Gay  and  Force  suggest  that  the  test  is  of 
presumptive  value  as  an  index  of  protection  against  typhoid  by 
vaccination.  Later  observations  by  them  confirm  this  view. 

Diagnosis. — The  diagnosis  of  typhoid  fever  in  the  living  subject  may 
be  made  either  by  the  isolation  and  identification  of  the  specific  organ- 
ism, Bacillus  typhosus,  or  by  the  demonstration  of  antibodies  specific 
for  this  organism  in  the  body  fluids  of  the  patient. 

(a)  BACTERIOLOGICAL  DIAGNOSIS. — 1.  Isolation  of  typhoid  bacilli 
from  the  blood  stream  and  from  rose  spots. 

1  Loc.  cit. 

2  University  of  California  Publications  in  Pathology,  1913,  ii,  No.  14;  Arch.  Int.  Med., 
1914,  xiii,  471. 


TYPHOID  BACILLUS  335 

Typhoid  bacilli  are  found  in  the  peripheral  blood  of  a  large  percen- 
tage of  typical  cases  of  typhoid  fever  during  the  first  week  of  the 
clinical  disease.  The  organisms  are  found  less  frequently  in  the  later 
stages.  The  statistics  reported  by  Coleman  and  Buxton,1  covering 
1137  cases,  show  this  clearly. 

Positive, 
Cases.  per  cent. 

First  week  of  clinical  disease 224  89 

Second  week  of  clinical  disease 484  73 

Third  week  of  clinical  disease        .......  268  60 

Fourth  week  of  clinical  disease 103  38 

Fifth  week  of  clinical  disease 58  26 

The  organisms  have  also  been  isolated  from  rose  spots  (which 
appear  as  a  rule  early  in  the  clinical  course  of  the  disease)  by  Richard- 
son and  others.  From  these  observations  typhoid  fever  may  be 
regarded  primarily  as  a  bacteremia.2  It  should  be  remembered,  how- 
ever, that  the  organisms  are  destroyed  in  the  blood  stream  by  specific 
lysins,  and  that  their  presence  in  the  circulating  fluids  of  the  body 
are  partly  caused  by  an  overflow  of  organisms  from  foci  in  the  spleen 
and  other  organisms. 

Method  of  Collecting  Blood. — The  skin  of  the  elbow  is  thoroughly 
cleansed  as  for  a  surgical  operation,  a  tourniquet  is  applied,  and  a 
large  hypodermic  needle  is  introduced  into  a  vein,  preferably  the 
median  basilic.  From  5  to  15  c.c.  of  blood  are  removed,  discharged 
at  once  into  a  flask  containing  150  to  250  c.c.  of  dextrose  broth  (0.1 
per  cent.),  and  mixed  thoroughly  before  clotting  takes  place.  This 
considerable  dilution  of  the  blood  is  important,  partly  because  clotting 
takes  place  more  slowly  and  thus  favors  the  escape  of  the  organisms 
into  the  broth,  and  also  because  it  dilutes  the  lysins  which  are  usually 
present  in  the  blood  of  typhoid  patients.  It  is  necessary  to  reduce 
the  concentration  of  lysins,  for  lysins  dissolve  typhoid  bacilli.  Incu- 
bation of  the  culture  at  37°  C.  for  twenty-four  hours  usually  results 
in  a  growth  of  bacteria  in  which  the  specific  organisms  are  present, 
either  alone  or  mixed  with  skin  cocci. 

Coleman  and  Buxton3  recommend  an  ox  bile  glycerin  peptone 
medium  for  the  isolation  of  typhoid  bacilli.  The  medium  as  prepared 
by  them  has  the  following  composition:  Ox  bile,  900  c.c.;  glycerin, 
100  c.c.;  peptone,  20  grams.  This  is  sterilized  in  the  autoclave  and 

1  Am.  Jour.  Med.  Sc.,  1907,  cxxxiii. 

2  Brion  and  Kayser,  Deut.  Arch.  f.  klin.  Med.,   1906,  Ixxxv,  552.      Coleman   and 
Buxton,  Jour.  Med.  Research,  1909,  xxi,  83.     Kolle  and  Hetsch,  Experimentelle  Bakt. 
und.  Infektionskrank.,  1911,  3ed.,  i,  250, 

3  Loc.  cit. 


336  THE  ALCALIGENES—DYSENTERY^TYPHOID 

distributed  in  flasks,  25  c.c.  to  a  flask.  The  ox  bile  prevents  the 
coagulation  of  the  blood.  Three  c.c.  of  blood,  according  to  the  Cole- 
man  technic,  are  added  to  the  flask  of  this  medium,  incubated  for 
eighteen  to  twenty-four  hours,  then  plated  out  on  agar.  Experience 
has  shown  that  larger  amounts  of  blood  are  more  satisfactory,  for 
it  has  been  found  that  not  infrequently  5  c.c.  of  blood  will  not  give 
a  growth  of  typhoid  bacilli,  whereas  10  c.c.  or,  better,  15  c.c.  will  give 
a  growth.  The  organisms  obtained  in  pure  culture  are  identified  by 
agglutination  with  a  known  specific  typhoid  serum  of  high  potency. 
Such  a  serum  used  in  high  dilution  reduces  the  possibility  of  "group 
agglutinins"  which  might  otherwise  give  an  erroneous  diagnosis. 
It  must  be  remembered  that  occasional  strains  of  typhoid  bacilli  are 
isolated  from  the  body  which  are  typical  culturally,  but  which  are 
non-agglutinable.  Frequently  a  few  successive  transfers  of  these 
organisms  on  artificial  media  will  restore  their  agglutinating  properties; 
occasionally,  however,  a  strain  is  met  with  which  will  not  agglutinate 
with  specific  typhoid  serum  even  after  long-continued  transfer  on 
artificial  media.  Mclntosh  and  McQueen1  have  found  that  at  least 
certain  strains  of  these  non-agglutinable  typhoid  bacilli  will  stimulate 
the  production  of  typical  typhoid  agglutinins  if  they  are  injected  into 
animals.  The  agglutinins  developed  in  these  animals  will  promptly 
clump  agglutinable  typhoid  bacilli,  but  will  not  agglutinate  the  non- 
agglutinable  strains  which  incited  the  production  of  these  agglutinins. 
These  non-agglutinable  strains,  however,  will  absorb  the  agglutimns 
apparently  as  readily  as  the  agglutinating  strairs.  Gay  and  Claypole2 
have  found  similarly  that  occasional  strains  of  typhoid  bacilli  isolated 
from  "typhoid  carrier"  rabbits  may  be  non-agglutinable.  They 
absorb  agglutinin,  however.  They  suggest  the  use  of  sera  obtained 
from  animals  immunized  with  cultures  of  typhoid  bacilli  grown  upon 
agar  containing  the  blood  of  man.  The  isolation  of  typhoid  bacilli 
from  the  blood  stream  and  their  identification  establishes  the  diag- 
nosis of  typhoid  fever  beyond  question  of  doubt. 

The  isolation  of  typhoid  bacilli  from  rose  spots  is  performed  in  essen- 
tially the  same  manner,  except  that  fluid  is  expressed  from  the  rose 
spot  after  the  skin  is  sterilized  over  it,  and  the  expressed  fluid  is  grown 
either  in  the  dextrose  broth  or  in  the  bile  medium.  Neufeld3  and 
Richardson4  have  successfully  isolated  typhoid  bacilli  from  the  roseola 

1  Jour.  Hyg.,  1914,  xiii,  409. 

2  Jour.  Am.  Med.  Assn.,  1913,  Ix,  1141;   Arch.  Int.  Med.,  1913,  xii,  613. 
'Ztschr.  f.  Hyg.,  1899,  xxx,  498. 

4  Philadelphia  Med.  Jour.,  March  3,  1900. 


TYPHOID  BACILLUS  337 

of  typhoid  fever  in  a  considerable  number  of  cases.  Thus,  Neufeld1 
obtained  cultures  in  13  of  14  cases  examined,  and  Richardson  obtained 
them  in  5  out  of  6  cases.  Both  Neufeld  and  Richardson  emphasize 
the  importance  of  incising  several  spots.  The  technic  developsd  by 
Richardson  is  as  follows:  the  skin  over  several  rose  spots  is  cleaned 
as  for  a  surgical  operation  and  then  frozen  by  a  spray  of  ethyl  chloride. 
This  procedure  drives  out  most  of  the  blood,  as  well  as  making  the 
operation  practically  painless.  A  small  incision  is  then  made  with 
a  sterile  knife  and  the  substance  of  the  rose  spot  is  removed  with  a 
small  skin  curette  and  at  once  placed  in  0.1  per  cent,  dextrose  broth, 
and  incubated  for  eighteen  to  twenty-four  hours.  The  identification 
of  the  bacilli  which  develop  in  the  broth  is  made  by  the  usual  cultural 
and  agglutination  reactions. 

2.  Isolation  of  Typhoid  Bacilli  from  the    Urine. — Typhoid  bacilli 
have  been  found  in  the  urine  in  from  25  to  35  per  cent,  of  the  cases 
examined.    Such  urines  frequently  contain  albumin.    The  organisms 
do  not  as  a  rule  appear  until  the  third  week  of  the  disease,  conse- 
quently their  isolation  is  of  comparatively  little  value  diagnostically, 
although  their  recognition  is  of  great  importance  for  the  prevention 
of  secondary  cases.    The  organisms  may  exist  in  the  urine  for  a  few 
weeks  after  recovery.    Rarely  they  persist  for  months  or  very  rarely 
for  years  after  recovery.     Frequently  their  presence  is  not  mani- 
fested by  clinical  symptoms,  but  occasionally  persistent  cystitis  may 
be  caused  by  their  continued  growth  in  the  urinary  bladder.    Usually 
the  bacilli  present  in  the  urine  are  found  in  pure  culture.    Occasionally 
colon  bacilli  are  found  either  in  association  with  typhoid  bacilli  or 
even  in  pure  culture  after  the  typhoid  bacilli  have  disappeared. 

3.  Isolation  of   Typhoid   Bacilli  from  Feces.— Typhoid  bacilli  are 
usually  found  in  pure  culture  or  nearly  pure  culture  in  the  blood,  and, 
if  the  proper  precautions  are  observed,  in  the  urine  as  well.    In  the 
feces,  on  the  contrary,  they  are  usually  in  the  minority  and  their 
isolation  presents  certain  difficulties.     It  has  been  claimed  by  many 
authorities  that  typhoid  bacilli  are  not  found  in  the  feces  in  demon- 
strable numbers,  at  least  until  about  the  middle  of  the  second  week. 
Klinger2  has  collected  statistics  from  812  contact  cases  which  indicate 
the  danger  of  infection  from  feces  even  before  the  development  of 
clinical  symptoms. 

1  Loc.  cit. 

2  Public  Health  Reports,  1911,  xxvi,  319. 

22 


338  THE  ALCALIGENES— DYSENTERY— TYPHOID 

SECONDARY  CASES  INFECTED  FROM  PRIMARY  CASES. 

First  week  of  incubation  period 33 

Second  week  of  incubation  period 150 

First  week  of  disease 1S7 

Second  week  of  disease 158 

Third  week  of  disease 116 

Fourth  week  of  disease 59 

Fifth  week  of  disease 34 

Sixth  week  of  disease 22 

Seventh  week  of  disease 14 

Eighth  week  of  disease 16 

Ninth  week  of  disease 15 

The  isolation  and  identification  of  typhoid  bacilli  from  the  feces 
is  by  no  means  proof  that  the  case  under  consideration  is  typhoid  fever; 
the  patient  may  be  a  carrier. 

Technic  of  Isolation  of  Typhoid  Bacilli  from  Feces. — A  thin  uniform 
emulsion  of  feces  suspected  to  contain  typhoid  bacilli  is  made  in  0.1 
per  cent,  dextrose  broth  and  incubated,  if  time  permits,  for  one  hour 
at  37°  C. 

The  emulsion  is  best  made  by  repeatedly  running  a  rather  heavy 
platinum  needle  through  the  fecal  mass  to  insure  a  representative 
sample.  The  process  is  continued  until  the  desired  density  of  bacteria 
in  the  broth  tube  is  attained.  Incubation  of  one  hour  permits  of  a 
slight  development  of  all  the  organisms;  it  particularly  acclimatizes 
the  typhoid  bacilli  to  artificial  media.  The  emulsion  is  then  spread 
with  a  bent  sterile  glass  rod  on  the  surface  of  Endo  medium  previously 
prepared  in  large  Petri  dishes.1  The  Petri  dishes  after  inoculation 
are  inverted  and  placed  in  the  incubator  at  37°  C.  and  examined  eigh- 
teen to  twenty-four  hours  later  for  clear,  colorless,  transparent  colonies 
which  rarely  attain  a  diameter  exceeding  2  mm.  These  colonies  are 
transferred  to  0.1  per  cent,  dextrose  broth  and  after  incubation  for 
eighteen  to  twenty-four  hours  at  37°  C.  are  mixed  with  a  high  potency 
antityphoid  serum  and  examined  for  agglutination. 

Rapid  Method  of  Isolating  Typhoid  Bacilli.2 — It  is  frequently  pos- 
sible to  identify  typhoid  bacilli  (and  paratyphoid  and  dysentery  bacilli 
as  well)  in  feces  within  twenty-four  hours  by  taking  advantage  of  the 
microscopic  agglutination  method  with  a  high  potency  serum  in  the 
following  manner:  Endo  plates  are  inoculated  as  indicated  above 
and  incubated  at  37°  C.  for  fifteen  to  eighteen  hours.  Typical  colonies 
are  removed  entire  to  small  test-tubes  containing  1  c.c.  of  0.1  per 
cent,  dextrose  broth  which  have  been  kept  at  incubator  temperature. 

1  For  preparation  and  use  of  the  Endo  medium,  see  page  201. 

2  Kendall  and  Day,  Jour.  Med.  Research,  1911,  xx,  95. 


TYPHOID  BACILLUS  339 

Incubation  of  these  infected  tubes  for  one  to  two  hours  almost  invari- 
ably gives  sufficient  numbers  of  organisms  to  make  a  microscopic 
agglutination.  A  confirmatory  cultural  diagnosis  may  be  obtained 
by  the  inoculation  of  small  tubes  of  semi-solid  media  and  milk  with 
the  remainder  of  the  troth  culture.  This  method  differs  from  the  one 
usually  employed  merely  in  the  small  amount  of  broth  used,  which 
requires  less  bacteria  to  produce  turbidity,  and  in  the  fact  that  the 
growth  is  practically  continuous  from  the  Endo  medium  to  the  tube, 
the  broth  being  warmed  to  the  body  temperature  at  the  start.  Taking 
advantage  of  these  factors  cuts  down  the  time  required  for  diagnosis 
nearly  twentv-four  hours. 

(6)  SEROLOGICAL  DIAGNOSIS. — The  blood  serum  of  patients  who 
have  recovered  from  a  typical  attack  of  typhoid  fever  contains  elements 
which  give  specific  reactions  with  the  typhoid  bacillus  or  its  products; 
of  these,  lysins,  agglutinins,  opsonins  and  precipitins  have  been 
carefully  studied.  The  method  of  fixation  of  complement  and  the 
ophthalmo  reaction  have  received  less  attention. 

The  lysins,  which  appear  early  in  the  course  of  the  disease,  dissolve 
typhoid  bacilli,  but  not  other  bacteria,  at  least  in  the  dilutions  ordi- 
narily used.  It  is  probable  that  the  lysins  not  only  dissolve  typhoid 
bacilli  in  vitro,  they  destroy  the  organisms  in  the  blood  stream  as 
well,1  liberating  endotoxins  which  play  a  prominent  part  in  the  produc- 
tion of  the  febrile  reaction. 

Agglutinins  are  formed  in  the  majority  of  cases,  which  will  clump 
typhoid  bacilli.  The  significance  of  agglutinins  in  the  typhoid  complex 
is  not  definitely  established. 

The  opsonic  index  of  the  serum  of  immunized  animals  and  of  clinical 
cases  of  typhoid  fever  in  man  appears  to  be  increased,  but  available 
methods  of  measuring  the  opsonic  index  do  not  furnish  information 
consistent  enough  to  warrant  definite  conclusions. 

The  reaction  of  fixation  of  complement  has  been  used  diagnostically 
in  a  limited  number  of  cases.  The  technical  skill  required  to  elicit 
satisfactory  results  has  doubtless  interfered  with  its  general  application. 

The  agglutination  reaction  is  by  far  the  most  commonly  used  anti- 
body reaction  employed  in  the  diagnosis  of  typhoid  fever. 

The  Widal  Reaction. — Historical. — Gruber  and  Durham  appear 
to  have  first  demonstrated  that  the  sera  of  animals  immunized  to 
typhoid  bacilli  would  agglutinate  the  typhoid  bacilli,  even  if  the 

1  Coleman  and  Buxton,  Medical  and  Surgical  Report  of  Bellevue  and  Allied  Hospitals, 
1909-10,  iv,  46. 


340  THE  ALCALIGENES— DYSENTERY— TYPHOID 

serum  were  diluted  many  times.  Griinbaum  and  later  Widal  applied 
this  principle  in  the  diagnosis  of  typhoid  fever.  It  is  now  recognized 
that  the  principle  involved  is  a  general  one  for  certain  kinds  of  bac- 
teria, and  the  Gruber-Durham-Gru'nbaum-Widal  reaction  is  used 
practically  in  the  diagnosis  of  several  diseases.  The  sera  of  such 
animals  frequently  contain  agglutinins  which  are  active  even  in 
dilutions  of  10^00  or  even  higher.  Specific  lysins  are  also  produced, 
which  in  dilutions  of  igg  to  nk)  will  dissolve  (and  kill)  typhoid  bacilli. 
1.  Collection  of  Blood  for  the  Agglutination  Test. — Dried  blood,  blood 
serum,  blister  fluid,  or  whole  blood  may  be  used  for  this  reaction. 

(a)  Dried  Blood. — A  generous  drop  of  blood  is  dropped  upon  a 
thin  sheet  of   aluminum   or  upon  clean,  glazed   paper,  and  allowed 
to  dry.    The  advantages  of  dried  blood  are:    (1)  it  is  easily  obtained 
by  making  a  puncture  in  the  ear  of  the  patient  and  collecting  a  drop 
of  blood;  (2)  it  does  not  lose  its  agglutinating  properties  readily; 
(3)  it    is    not    readily    contaminated;  and    (4)  the    blood    may    be 
removed  quantitatively  after  it  is  dried   (scaled  off),  weighed  and 
then  diluted  to  the  desired  degree  as  accurately  as  blood  serum.    The 
disadvantages  are:    (1)  flies  will  readily  remove  a  film  of  dried  blood; 
and  (2)  typhoid  bacilli  are  rarely  found  in  blood  clots.     There  is, 
however,  very  little  danger  of  spreading  typhoid  in  this  way.     In 
practice  dried  blood  is  diluted  with  physiological  normal  saline  solu- 
tion to  a  pale  rose  color,  which  corresponds  to  a  dilution  of  1  to  20. 
This  dilution  is  somewhat  inaccurate  and  anemic  bloods  introduce  a 
disturbing  factor.     This  method  of  dilution,  however,  is  sufficiently 
accurate  for  all  except  unusual  cases,  and  it  is  a  method  generally 
used  in  routine  board  of  health  examinations. 

(b)  Blood  Serum. — A  few  drops  of  blood  are  collected  in  a  capillary 
pipette  or  small  test-tube  and  allowed  to  clot.    The  serum  is  removed 
and  diluted  accurately  with  salt  solution.    The  advantages  are:     (1) 
the  accuracy  with  which  dilution  may  be  made;  and  (2)  the  ease 
with  which  serum  is  obtained.     The  disadvantages  are:     (1)  that 
blood  serum  is  readily  contaminated;  and  (2)  it  does  not  keep  well, 
it  deteriorates.    Blood  serum  is  the  best  for  accurate  work. 

(c)  Blister  Fluid. — This  possesses  no  advantages  over  blood  serum. 
It  is  somewhat  more  difficult  to  obtain  and  probably  somewhat  less 
accurate  than  blood  serum. 

(d)  Whole  Blood.— Aside  from  clotting,  whole  blood  is  as  reliable 
as  blood  serum,  so  far  as  accuracy  of  dilution  and  potency  of  agglu- 
tinins is  concerned.     It  must  be  remembered,  however,  that  the  red 


TYPHOID  BACILLUS  341 

blood  cells  appear  in  the  field  viewed  under  the  microscope.  Fresh 
whole  blood  presents  one  great  disadvantage — the  fibrin  in  it  may 
cause  a  pseudoagglutination,  for  the  fibrin  network  that  forms  as 
coagulation  proceeds  entangles  typhoid  bacilli  in  its  meshes,  giving 
the  appearance  of  a  true  agglutination.  Whole  blood  can  be  con- 
veniently drawn  into  a  blood-counting  pipette  and  diluted  accurately 
and  immediately. 

The  Culture  to  be  Used. — Old  stock  cultures  of  typhoid  bacilli  usually 
give  the  best  results.  Freshly  isolated  cultures  not  infrequently  agglu- 
tinate less  readily  than  those  which  have  been  on  artificial  media  for 
some  time.  The  organisms  should  be  grown  in  0.1  per  cent,  dextrose 
broth  for  eighteen  hours  at  30°  to  32°  C.  It  has  been  found  that 
typhoid  bacilli  grown  at  this  temperature  agglutinate  somewhat 
better  than  those  grown  at  37°  C.  Killed  cultures  are  frequently 
used,  but  the  results  obtained  are  somewhat  less  accurate  than  those 
with  living  cultures.  In  rare  instances  it  has  been  found  that  killed 
cultures  will  agglutinate  with  typhoid  sera  at  45°  C.  when  living 
cultures  fail  to  agglutinate.  Controls  must  always  be  made:  the 
typhoid  culture  is  diluted  with  an  equal  volume  of  salt  solution. 
Spontaneous  agglutination  sometimes  takes  place  when  no  serum  is 
present.  This  is  shown  in  the  control  and  at  once  invalidates  the 
agglutination  which  may  be  obtained  with  the  serum. 

Technic  of  Test. — (A)  Microscopic  Method. — Dried  blood,  blood 
serum,  blister  fluid,  or  whole  blood  is  diluted  1  to  20  with  physiological 
salt  solution.  A  loopful  of  this  diluted  fluid  is  mixed  intimately  with 
a  loopful  of  typhoid  broth  culture  on  a  coverglass  and  suspended  in 
a  hanging  drop  slide  ringed  with  vaseline  to  prevent  evaporation. 
The  final  dilution  of  the  blood  is  1  to  40  by  this  procedure.  A  control 
is  made  using  a  loopful  of  salt  solution  and  a  loopful  of  typhoid  culture 
prepared  in  the  same  manner.  Both  the  serum  and  the  control  are 
kept  at  room  temperature.  A  preliminary  examination  should  show 
actively  motile  bacteria  in  the  control  preparation  and  usually  actively 
motile  bacteria  in  the  serum  preparation.  It  sometimes  happens  that 
agglutination  takes  place  in  the  serum  preparation  almost  immediately. 
If  the  preliminary  examination  is  satisfactory,  the  final  examination 
is  made  at  the  end  of  an  hour.  Both  preparations  are  examined  and 
the  controls  should  show  actively  motile  unclumped  organisms.  A 
positive  agglutination  is  recorded  if  the  control  is  as  stated  and  the 
organisms  in  the  serum  preparation  are  non-motile  and  gathered 
together  in  clumps  with  few  or  no  free-swimming  bacteria  between 
the  clumps. 


342  THE  ALCALIGENES— DYSENTERY— TYPHOID 

(B)  Macroscopic  Method. — Various  dilutions  of  serum  are  placed 
in  small  sterile  test-tubes,  1  c.c.  in  each  test-tube.  As  a  routine,  a 
dilution  of  1  to  20  is  used,  but  a  series  of  dilutions  up  to  the  limits 
of  the  serum  are  frequently  made.  To  each  tube  is  added  1  c.c.  of  a 
broth  culture  of  typhoid  bacilli.  A  control  is  made  by  adding  1  c.c. 
of  a  broth  culture  of  typhoid  bacilli  to  1  c.c.  of  salt  solution.  These 
mixtures  are  respectively  shaken  and  incubated  together  with  the 
control  at  37°  C.  for  two  hours,  then  they  are  placed  in  the  ice-box, 
and  examined  eighteen  to  twenty-four  hours  later.  A  positive  agglu- 
tination is  indicated  when  the  supernatant  fluid  of  the  serum  typhoid 
mixtures  is  clear,  while  the  control  containing  no  serum  remains 
uniformly  cloudy. 

The  microscopic  method  is  much  more  rapid  than  the  macroscopic 
method  and  is  sufficiently  accurate  for  ordinary  purposes.  The  macro- 
scopic method  requires  a  much  longer  time,  but  it  is  more  accurate, 
for  the  dilutions  can  be  made  carefully  with  graduated  pipettes. 

Discussion. — Available  statistics  show  that  about  20  per  cent,  of 
typhoid  patients  exhibit  a  positive  agglutination  reaction  at  the  end 
of  the  first  week;  60  per  cent,  at  the  end  of  the  second  week;  80  per 
cent,  at  the  end  of  the  third  week;  and  90  per  cent,  at  the  end  of  the 
fourth  week.  These  agglutinins  persist;  about  75  per  cent,  of  all 
patients  exhibit  a  positive  agglutination  after  two  months.  Occa- 
sionally agglutinins  may  persist  for  several  years.1  The  amount  of 
agglutination  present,  as  indicated  by  the  degree  of  dilution  which 
will  still  clump  typhoid  bacilli,  has  no  known  relationship  to  the 
severity  of  the  attack.  An  occasional  mild  case  of  typhoid  may  be 
accompanied  by  the  appearance  of  agglutinins  of  great  potency; 
severe  attacks  may  exhibit  little  or  no  agglutinin  in  the  blood.  Occa- 
sionally, agglutinins  are  not  demonstrable  in  the  blood  serum  of 
undoubted  cases  of  typhoid  fever.  This  has  been  found  to  be  the  case 
by  Moreschi2  in  several  cases  of  chronic  leukemia.  Moreschi3  has  made 
the  interesting  observation  that  even  the  vaccination  of  these  leukemics 
with  killed  cultures  of  typhoid  bacilli  may  not  lead  to  the  development 
of  agglutinins.  In  icterus  an  agglutination  is  not  infrequently  encoun- 
tered even  if  the  serum  is  highly  diluted.  It  is  very  probable  that  at 
least  some  of  these  cases  are  typhoid  carriers,  having  typhoid  bacilli  in 
the  gall-bladder.  They  may  be  ambulatory  cases.  It  has  been  claimed 

1  An  initial  negative  reaction  (first  week)  followed  by  a  positive  reaction  is  conclusive. 
It  rules  out  the  possibility  of  persistent  agglutinins  from  previous  cases,  and  those  due 
to  protective  vaccination. 

2  Ztschr.  f.  Immunitatsforsch.,  1914,  xxi,  410.  3  Loc.  cit. 


TYPHOID  BACILLUS  343 

by  some  observers  that  the  agglutination  seen  in  icteric  patients 
is  due  to  bile  in  the  blood  stream.  This,  however,  has  not  been 
proven.  A  negative  agglutination,  when  the  clinical  symptoms  suggest 
typhoid  fever,  should  suggest  the  possibility  of  a  paratyphoid  infection. 

Ophthalmo  Reaction. — Chantemesse1  has  found  that  an  ophthalmo 
reaction  may  be  elicited  in  typhoid  patients  similar  to  that  produced 
by  the  introduction  of  tuberculin  in  the  eye  of  the  tuberculous  patient. 
Broth  cultures  of  typhoid  bacilli  are  precipitated  with  alcohol;  the 
precipitate  is  dried  and  pulverized;  ^  milligram  of  the  powder  is 
dissolved  in  a  few  drops  of  sterile  saline  solution  and  introduced  into 
the  eye.  A  transient  redness  with  a  flow  of  tears  occurs  in  normal 
individuals;  a  severe  reaction  (even  accompanied  by  a  serofibrinous 
exudate  in  unusual  cases),  which  reaches  its  maximum  intensity 
within  twelve  hours,  is  elicited  in  typhoid  patients,  and,  occasionally, 
in  individuals  who  have  recovered  from  the  disease.  The  diagnostic 
value  of  the  reaction  is  as  yet  undetermined. 

Dissemination  and  Prophylaxis. — The  disease  typhoid  fever  occurs 
only  by  transmission  of  typhoid  bacilli  directly  or  indirectly  from 
preexisting  cases.  The  disease  is  acquired  only  by  the  ingestion  of 
the  specific  organisms,  and  infection  by  any  other  channel  than  the 
alimentary  canal  has  not  so  far  been  satisfactorily  demonstrated. 

Prophylactic  measures,  therefore,  should  begin  with  the  isolation 
of  the  patient  and  disinfection  of  all  excreta  and  all  utensils  which 
have  been  in  contact  with  the  patient.  The  organism  may  occur  in 
the  fecal  discharges  of  patients  before  clinical  symptoms  develop,  in 
patients  recently  recovered  from  the  disease,  in  carriers  (which  number 
about  2  per  cent,  of  all  cases  diagnosed),  and  probably  in  a  relatively 
few  individuals  in  whom  the  organism  may  gain  a  temporary  foothold 
without  producing  symptoms.  The  bacilli  may  be  transmitted  to 
others  by  the  hands  of  those  who  care  for  the  patients,  and  the  hands 
of  carriers.  Fecal  matter  containing  typhoid  bacilli  may  be  trans- 
ferred by  flies,  by  water,  through  milk,  and  perhaps  by  vegetables 
which  are  eaten  uncooked.  The  water  in  which  typhoid  patients  have 
bathed  is  frequently  grossly  contaminated  with  the  organisms.  Rarely, 
wells  and  water  supplies  are  contaminated  by  urinary  typhoid  car- 
riers, in  which  event  the  colon  bacillus,  which  is  ordinarily  relied  upon 
for  evidence  of  contamination,  may  be  absent.  A  thorough  disin- 
fection of  excreta  including  urine  will  prevent  spread  of  the  disease 
from  known  cases. 

1 IV.  International  Cong,  of  Demog.  and  Hyg.,  Berlin,  September  26,  1907. 


344  THE   ALCALIGENES— DYSENTERY— TYPHOID 

THE   PARATYPHOID    GROUP. 

There  is  a  group  of  closely  related  bacilli  which  exhibit  cultural 
and  pathogenic  characters  intermediate  between  those  of  the  typhoid, 
dysentery  and  colon  groups  of  bacteria,  respectively.  These  organ- 
isms are  variously  known  as  the  hog  cholera,  Salmonella,  Gartner, 
enteritidis,  intermediate,  paracolon  or  paratyphoid  group. 

Smith  and  Salmon1  isolated  the  type  organism  of  the  group  from 
the  intestinal  contents  of  swine  infected  with  hog  cholera.  They 
named  their  organism  the  hog  cholera  bacillus.2  Three  years  later 
Gartner3  described  an  organism,  B.  enteritidis,  recovered  by  him  both 
from  the  spleen  and  blood  of  a  fatal  case  of  meat-poisoning,  and  from 
the  suspected  meat  (beef)  itself.  Numerous  epidemics  of  meat  poison- 
ing4 have  been  studied  bacteriologically  during  the  years  following 
Gartner's  discovery,  and  very  similar,  if  not  identical,  bacilli  have 
been  recovered  from  many  of  the  patients. 

In  1893  Smith  and  Moore5  made  the  important  observation  that 
organisms  culturally  indistinguishable  from  the  hog  cholera  bacillus 
could  be  isolated  not  infrequently  from  the  intestinal  contents  of 
normal  cattle,  swine,  sheep,  cats  and  dogs.  The  significance  of  this 
discovery  from  the  view-point  of  meat  poisoning  was  not  understood 
at  that  time. 

In  1896  Achard  and  Bensaude6  described  paratyphoid  fever  and 
outlined  the  essential  clinical  and  bacteriological  diagnostic  differences 
between  this  disease  and  typhoid  fever.  They  obtained  paratyphoid 
bacilli  from  the  urine  and  blood  stream  of  several  cases,  and  recovered 
the  organism  from  a  secondary  purulent  arthritis  in  one  of  them  as 
as  well.  Schottmuller7  also  obtained  cultures  of  paratyphoid  bacilli 
both  from  the  feces  and  the  blood  stream  of  several  cases  of  para- 
typhoid fever.  Brion  and  Kayser8  separated  these  organisms  into  two 
types:  B.  paratyphosus  alpha,  which  produced  a  slight  permanent 
acidity  in  litmus  milk  and  gave  an  "invisible"  growth  on  potato 

1  Ann.  Rep.  United  States  Bur.  Animal  Ind.,  1885,  vol.  ii. 

2  A  year  earlier  Klein   (Virchows  Arch.,   1884,  xcv,  468)   obtained  a  bacillus  from 
diseased  swine  which  he  regarded  as  the  causative  factor  of  hog  cholera,  but  his  organism 
produced  spores,  which  at  once  distinguished  it  from  the  paratyphoid  type.     Neither  the 
Klein  bacillus  nor  the  Smith-Salmon  bacillus  cause  hog  cholera;  a  filterable  virus  is  the 
probable  infecting  agent. 

3  Correspondz.-Blatt  des  allgem.  arztl.  Vereins  von  Thuringen,  1888,  No.  9. 

4  Not  to  be  confused  with  botulismus  (see  B.  botulinus). 

5  Additional  investigations  concerning  swine  diseases,  Washington,  D.  C.,  1893. 

6  Soc.  Med.  des  Hop.  de  Paris,  1896,  3d  Sens,  xiii,  679. 

7  Deutsch.  med.  Wchnschr.,  1900,  p.  511. 

8  Munchen.  med.  Wchnschr.,  1902,  p.  611. 


THE  PARATYPHOID  GROUP  345 

like  the  typhoid  bacillus;  and  B.  paratyphosus  beta,  which  produced 
an  initial  acidity  in  litmus  milk  followed  by  a  progressively  alkaline 
reaction.  These  observations,  both  clinical  and  bacteriological,  have 
been  confirmed  by  later  investigations. 

Morphology. — The  members  of  the  intermediate  group  are  indistin- 
guishable morphologically.  They  are  rod-shaped  bacilli  with  rounded 
ends,  measuring  from  0.8  to  1  micron  in  diameter,  and  1.5  to  3.5 
microns  in  length,  occurring  singly  or  in  pairs,  seldom  in  chains.  In 
actively-growing  cultures  the  organisms  may  be  short,  almost  ovoid. 
In  old  cultures  the  organisms  may  be  elongated;  filamentous  forms 
are  more  commonly  seen  in  old  gelatin  cultures.  The  members  of  the 
group  are  actively  motile  and  possess  from  four  to  twelve  peritrichic 
flagella.  Motility  is  greater  in  dextrose  broth  than  in  plain  broth; 
this  is  particularly  the  case  in  young  cultures.  The  organisms  form 
no  spores  and  appear  to  possess  no  capsules.  They  stain  readily  with 
ordinary  anilin  dyes;  occasionally  organisms  from  cultures  several 
days  old  exhibit  a  tendency  toward  bipolar  staining.  They  are  Gram- 
negative. 

Isolation  and  Culture. — The  organisms  of  the  paratyphoid  group  grow 
readily  upon  ordinary  artificial  media,  B.  paratyphosus  alpha  somewhat 
less  luxuriantly  than  the  remaining  members.  The  colonies  produced 
on  agar  after  eighteen  hours'  incubation  at  37°  C.  resemble  those 
of  the  typhoid-dysentery  group — small,  round,  and  transparent — 
measuring  from  1  to  3  mm.  in  diameter.  On  Endo  medium  the  colonies, 
like  those  of  B.  typhosus  and  the  dysentery  bacilli,  are  clear  and 
colorless  and  somewhat  smaller  than  those  developing  upon  plain 
agar.  They  usually  measure  from  0.75  to  2  mm.  in  diameter.  The 
organisms  grow  well  in  gelatin,  but  do  not  cause  liquefaction.  They 
produce  acid  and  gas  in  dextrose  and  mannite;  lactose  and  saccharose 
are  not  fermented. 

Milk. — Plain  milk  is  not  coagulated.  All  the  members  of  the  group 
except  B.  paratyphosus  alpha  cause  a  slow  change  in  this  medium, 
which  becomes  thin,  brownish,  and  almost  opalescent  after  two  or 
more  weeks'  incubation.  In  litmus  milk  the  cream  ring  is  colored 
a  deep  blue-green,  which  is  so  constant  as  to  be  suggestive  diagnos- 
tically.  B.  paratyphosus  alpha  produces  a  slight  acidity  which  is 
permanent;  the  milk  assumes  a  lilac  color.  B.  paratyphosus  beta 
and  other  members  of  the  group  produce  a  transient  acidity1  which 

1  For  an  explanation  of  the  phenomenon,  see  page  222. 


346  THE  ALCALIGENES— DYSENTERY— TYPHOID 

is  followed  by  a  progressive  alkalinity,  associated  with  the  liberation 
of  small  amounts  of  ammonia.1 

All  members  of  the  intermediate  group  produce  considerable  tur- 
bidity in  plain  and  sugar  broths.  A  pellicle  may  develop  in  plain 
broth  after  several  days'  incubation.  Potato:  B.  paratyphosus 
alpha  grows  much  like  the  typhoid  bacillus  on  potato;  the  growth  is 
nearly  invisible  on  acid  potato,  but  comparatively  luxuriant.  On 
alkaline  potato  the  growth  is  brownish.  B.  paratyphosus  beta  pro- 
duces a  brownish  growth  even  on  slightly  acid  potato,  which  resembles 
that  characteristic  of  B.  coli. 

The  members  of  the  intermediate  group  are  all  aerobic,  facultatively 
anaerobic.  The  minimum  temperature  of  growth  is  about  6°  to  8°  C., 
the  optimum  37°  C.,  and  growth  ceases  at  approximately  44°  C.  The 
resistance  of  the  members  of  the  intermediate  group  to  environmental 
conditions,  drying  and  to  chemicals  is  similar  to  that  of  the  typhoid 
bacillus.  They  are,  however,  somewhat  more  resistant  to  heat;  an 
exposure  of  fifteen  minutes  at  70°  C.  or  of  five  minutes  at  75°  C., 
kills  the  bacilli.  This  is  a  point  of  importance  in  meats  infected 
with  the  organisms;  temperatures  lower  than  75°  C.  in  the  centre  of 
the  meat  can  not  be  relied  upon  to  remove  danger  of  infection.  Higher 
temperatures,  100°  C.,  are  preferable  to  remove  all  danger  from  the 
poisonous  substances  of  the  bacilli,  which  are  not  destroyed  by  gastro- 
intestinal digestion. 

Products  of  Growth. — (a)  Chemical. — Paratyphoid  bacilli  are  rather 
more  active  proteolytically  than  typhoid  and  dysentery  bacilli,  but 
they  produce  neither  phenols  nor  indol.2  Dextrose  and  mannite  are 
fermented  with  the  formation  of  carbon  dioxide  and  hydrogen,  lactic 
acid,  and  smaller  amounts  of  acetic  and  formic  acids.  Lactose  and 
saccharose  are  not  fermented.  Numerous  attempts  have  been  made 
to  classify  the  paratyphoid  bacilli  into  several  varieties  upon  the  basis 
of  the  fermentation  of  carbohydrates  other  than  those  mentioned 
above,  but  the  lack  of  agreement  has  proved  an  insurmountable 
obstacle  to  their  general  acceptance. 

(b)  Enzymes. — The  members  of  the  paratyphoid  group  do  not  pro- 
duce soluble  proteolytic  ferments,  and  they  do  not  liquefy  coagulated 
blood  serum,  gelatin,  fibrin  or  egg  albumen.  Neither  lipolytic  nor 
amylolytic  enzymes  have  been  demonstrated  in  cultures  of  these 
organisms. 

1  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Soc.,  1914,  xxxvi,  1943. 

2  Ibid.,  1913,  xxxv,  1221. 


THE  PARATYPHOID  GROUP  34? 

(c)  Toxins. — Soluble  toxins  have  not  been  demonstrated  in  cul- 
tures of  paratyphoid  bacilli.  Cathcart1  and  Franchetti2  have  shown 
that  minute  amounts  of  autoly sates  of  the  organisms  are  rapidly* fatal 
to  rabbits  and  other  small  laboratory  animals.  According  to  Cath- 
cart,3 the  poisonous  substance  (endotoxin)  liberated  from  the  organ- 
isms during  autolysis  is  relatively  thermostabile ;  a  brief  exposure  of 
it  to  100°  C.  does  not  completely  destroy  its  potency. 

Classification  and  Identification  of  the  Paratyphoid  Group. — It  is  pos- 
sible to  divide  the  Paratyphoid  Group  into  two  distinct  types  by  their 
reaction  in  milk:  the  alpha  type,  of  which  several  strains  have  been 
described,  differing  somewhat  in  their  serological  reactions;  and  the 
beta  type.  The  former  appears  to  be  limited  to  man,  but  the  latter 
comprises  organisms  which  are  rather  widely  distributed  not  only 
in  man  but  in  the  lower  animals  as  well.  The  better  known  strains 
of  the  beta  type  comprise  not  only  B.  paratyphosus  beta,  B.  enteritidis 
and  the  hog  cholera  bacillus  (B.  choleras  suis,  B.  suipestifer)  mentioned 
above,  but  B.  psittacosis,  obtained  from  infectious  enteritis  of  parrots, 
which  produces  a  pneumonic  infection  in  man.4  B.  icteroides,  San- 
arelli,  originally  supposed  to  cause  yellow  fever,  but  now  known  to 
be  indistinguishable  from  the  hog  cholera  bacillus,  the  Danysz  bacillus 
of  rat  plague,  and  B.  typhi  murium,  Loffler,  obtained  from  epizootics 
of  rodents,  B.  sertrycke,  de  Nobele,  and  B.  moorseele,  van  Ermengem, 
from  epidemics  of  meat  poisoning,  and  B.  morbificans  bo  vis,  Basenau, 
isolated  from  a  diseased  cow,  all  belong  to  the  same  group.  They 
possess  in  common  cultural  characteristics  which  differ  somewhat 
quantitatively,  but  not  qualitatively.  Bainbridge  and  O'Brien5  have 
attempted  to  classify  the  organisms  by  agglutination  and  absorption 
tests;  they  recognize  four  groups  as  follows:  (1)  B.  paratyphosus 
alpha;  (2)  B.  paratyphosus  beta;  (3)  B.  suipestifer  (hog  cholera  bacil- 
lus), including  B.  psittacosis,  B.  sertrycke  and  some  strains  of  B. 
typhi  murium;  (4)  B.  enteritidis,  including  the  Danysz  bacillus,  B. 
morbificans  bovis,  and  some  strains  of  B.  typhi  murium.  This  clas- 
sification, if  substantiated,  possesses  the  advantage  of  separating 
those  organisms  which  cause  paratyphoid  fever,  the  alpha  and  beta 
types,  from  the  bacilli  more  commonly  associated  with  the  lower 

1  Jour.  Hyg.,  1906,  vi,  112. 

2  Ztschr.  f.  Hyg.,  1908,  Ix,  127. 

3  Loc.  cit. 

4  Nocard,  Conseil  d'hygiene  pub.  et  Salubrite  du  Dept.  du  Seine,  S6ance,  March  24, 
1893. 

5  Jour.  Hyg.,  1911,  xi,  68. 


348  THE  ALCALIGENES— DYSENTERY— TYPHOID 

animals,  of  which  the  hog  cholera  bacillus  and  B.  enteritidis  are  the 
types.  This  classification  has  not  been  universally  accepted,  how- 
ever. Doubtless  the  multiplicity  of  strains  which  have  received  the 
same  name  has  led  to  confusion  in  standard  type  organisms  which  are 
especially  essential  in  this  line  of  investigation.  It  is  not  an  assured 
fact  that  the  paratyphoid  bacilli,  alpha  and  beta,  are  restricted  to  the 
production  of  paratyphoid  fever  in  man;  nor  can  it  be  stated  definitely 
that  B.  enteritidis  and  the  hog  cholera  bacillus  consistently  cause 
meat  poisoning.  Available  information  suggests  that  occasionally 
the  choleraic  symptoms  of  meat  poisoning  may  be  elicited  by  para- 
typhoid bacilli,  and  that  the  symptoms  of  paratyphoid  fever  may 
follow  infection  with  B.  enteritidis  or  B.  suipestifer. 

Pathogenesis. — Animal. — The  members  of  the  Paratyphoid  Group 
are,  as  a  rule,  very  pathogenic  for  small  laboratory  animals.  The  intra- 
peritoneal  injection  of  very  minute  amounts  of  bacilli  usually  causes 
acute  death  in  guinea-pigs  and  mice.  Rats  are  somewhat  more 
resistant.  B.  typhi  murium  and  other  arat  viruses"  produce  a  fatal 
enteritis  in  mice  and  rats ;  the  bacilli  are  present  not  only  in  the  intes- 
tinal contents,  they  may  be  obtained  from  the  tissues  and  organs  post- 
mortem as  well.  Bacilli  belonging  to  the  Paratyphoid  Group  have 
been  isolated  from  epizootics  and  sporadic  cases  of  enteritis  in  cattle, 
parrots,  and  rodents.  The  organisms  appear  to  be  widely  distributed 
among  the  lower  animals. 

Human. — Three  types  of  disease  are  produced  in  man  by  the  bac- 
teria of  the  paratyphoid  group:  (a)  meat  poisoning:  the  symptoms 
are  choleraic  in  character,  and  they  may  be  severe  enough  to  be 
confused  with  true  cholera;1  infection  usually  follows  the  ingest  ion 
of  imperfectly  cooked  beef  or  pork  contaminated  with  B.  enteritidis 
or  the  hog  cholera  bacillus.  Somewhat  similar  symptoms  have 
resulted  from  the  accidental  ingestion  of  the  "rat  virus"  of  Danysz 
and  others;2  (b)  paratyphoid  fever,  a  disease  clinically  resembling 
mild  typhoid  fever,  usually  caused  by  B.  paratyphosus  alpha  or  B. 
paratyphosus  beta;  (c)  a  rare  type  of  disease,  pneumonic  in  character, 
produced  by  B.  psittacosis,  which  produces  an  epizootic  disease  among 
parrots. 

(a)  Meat  Poisoning. — The  disease  is  more  prevalent  in  summer  and 
fall  than  it  is  in  winter  and  spring,  probably  due  in  part  to  decreased 

1  Hetsch,  Klin.  Jahrb.,  1907,  xvi,  267. 

2  Mayer,   Miinchen.    med.    Wchnschr.,   1906,   No.  47;    Shibayama,   Miinchen.   med. 
Wchnschr.,  1907,  979. 


THE  PARATYPHOID  GROUP  349 

efficiency  of  refrigeration  of  meats  in  the  warmer  months.  The  incu- 
bation period  may  be  as  brief  as  four  to  six  hours,  or  as  long  as  twenty- 
four  to  seventy-two  hours  after  ingestion  of  the  infected  food.  The 
initial  symptoms  are  usually  a  severe  headache  and  chill,  rapidly 
followed  by  acute  gastro-intestinal  disturbances,  dizziness,  nausea 
and  vomiting,  abdominal  pain  and  diarrhea.  Nervous  symptoms  and 
marked  restlessness  are  characteristic  of  the  severe  and  fatal  cases. 
Usually  the  symptoms  and  fever  abate  within  a  week;  they  may 
persist  for  several  weeks.  The  mortality  is,  as  a  rule,  low,  averaging 
from  1  to  2  per  cent.  The  conspicuous  lesion  observed  at  autopsy 
is  an  intense  hyperemia  of  the  gastro-intestinal  mucosa,  usually  with- 
out noteworthy  involvement  of  Peyer's  patches.  Fatty  degeneration 
of  the  liver  is  common.  Bacilli  (usually  B.  enteritidis  or  B.  cholerae 
suis,1  less  commonly  B.  paratyphosus  beta)  may  be  isolated  from  the 
feces  and  blood  stream  in  many  of  the  acute  cases  during  the  first 
few  days  of  the  disease.  They  are  almost  invariably  recovered  from 
the  heart  blood  and  spleen  at  autopsy.  Serum  reactions,  especially 
specific  agglutinins,  may  be  demonstrated  at  the  end  of  the  first  week 
in  many  but  not  all  cases. 

An  epidemic  of  meat  poisoning  is  characterized  by  the  sudden,  prac- 
tically simultaneous  onset  of  symptoms  in  those  who  have  eaten  the 
contaminated  food,  and  the  limitation  of  the  disease  to  the  primary 
cases.  Secondary  infection  is  uncommon.  It  should  be  remembered 
that  not  all  epidemics  of  meat  poisoning  are  caused  by  members  of 
the  paratyphoid  group  of  bacteria. 

Distribution  of  Organisms. — The  hog  cholera  bacillus  (B.  cholera? 
suis,  B.  suipestifer)  is  frequently  found  in  the  intestinal  tracts  of 
swine,  rats  and  mice;  probably  somewhat  less  commonly  in  cattle. 
B.  enteritidis  is  a  frequent  inhabitant  of  the  intestinal  contents  of 
rats  and  mice,  and  relatively  uncommon  in  healthy  cattle.2  It  is 
suspected  that  a  postmortem  infection  of  beef  is  more  common  than 
an  antemortem  invasion;  this  is  reasonably  suggested  by  the  wide 
distribution  of  rats  and  mice  in  slaughter  houses.  The  organisms 
possess  the  somewhat  unusual  property  of  rapidly  diffusing  them- 
selves through  the  substance  of  meat  after  they  have  been  distributed 
on  the  surface  of  it  by  careless  handling.  Unless  infected  meat  is 
thoroughly  cooked,  the  organisms  are  not  killed,  and  they  may  not 
be  even  weakened  if  the  degree  of  heat  and  time  of  exposure  is  insuffi- 

1  Bainbridge,  Lancet,  March  16,  23,  30,  1912. 

2  Ibid. 


350  THE  ALCALIGENES— DYSENTERY— TYPHOID 

cient.  The  endotoxins  of  the  bacilli,  furthermore,  are  relatively 
thermostabile.  Thorough  cooking  of  such  meat  is  essential  to  insure 
safety. 

(b)  Paratyphoid  Fewr. — Bacteriologically,  paratyphoid  fever  may 
be  caused  either  by  B.  paratyphosus  alpha  or  B.  paratyphosus  beta. 
Clinically  there  is  little  or  no  difference  between  the  two  infections. 
According  to  Bainbridge,1  paratyphoid  fever  in  Asia,  particularly 
in  India,  is  more  frequently  an  infection  with  the  alpha  organism;  in 
Europe  the  beta  organism  is  much  more  frequently  reported.  Both 
types  are  found  in  the  United  States.2  The  organisms  are  occasionally 
found  in  the  intestinal  contents  and  feces  of  young  children  and  adults 
who  give  no  history  of  infection. 

The  incubation  period  of  paratyphoid  fever  varies  from  eight  to 
twenty  days;  the  average  is  about  two  weeks.  The  onset  is  gradual; 
the  usual  prodromal  symptoms  are  severe  head-  and  backache,  malaise 
and  anorexia.  Bronchitis  and  sore  throat  are  common.  There  may 
be  an  initial  chill,  then  the  temperature  rises  rather  rapidly  to  a  maxi- 
mum of  103°  to  105°  C.;  after  the  fifth  to  the  seventh  day  it  falls 
slowly;  it  is  normal  by  the  end  of  the  second  week.  Rose  spots  are 
occasionally  seen  early  in  the  disease.  Less  commonly  acute  gastro- 
enteric  symptoms,  resembling  those  of  meat  poisoning,  complicate  the 
clinical  picture.  Paratyphoid  fever  is  a  bacteremia,  very  similar  to 
typhoid  fever  in  this  respect.  The  mortality  is  low,  averaging  from 
1  to  2  per  cent,  of  all  cases.  The  lesions  observed  postmortem  are 
intense  hyperemia  of  the  gastro-intestinal  tract,  usually  with  superficial 
ulcerations  in  the  ileum  and  cecum,  not  necessarily,  however,  involv- 
ing Peyer's  patches.  Acute  splenic  tumor  is  usually  not  a  feature  of 
paratyphoid  infections.  The  bacilli  may  be  isolated  from  the  heart 
blood  and  visceral  organs. 

Bacterial  Diagnosis. — (a)  Isolation  of  Bacilli. — Blood  cultures  made 
during  the  first  week  are  frequently  positive.  The  organisms  are 
usually  present  in  the  feces,  occasionally  in  the  urine.  The  identifi- 
cation of  the  bacilli  depends  upon  the  cultural  characters  outlined 
above;  gas  production  in  dextrose  and  mannite,  no  liquefaction  of 
gelatin,  and  a  permanent  acidity  in  litmus  milk  (alpha  type)  or  a 
transient  acidity  followed  by  a  progressively  alkaline  reaction  in  this 

1  Loc.  cit. 

2Gwyn,  Bull.  Johns  Hopkins  Hospital,  1898,  vol.  ix.  Gushing,  ibid.,  1900,  vol.  xi; 
Buxton  and  Coleman,  Proc.  Path.  Soc.  New  York,  February,  1902;  Proescher  and 
Roddy,  Jour.  Am.  Med.  Assn.,  1909,  lii,  No.  6;  Kendall,  Bagg  and  Day,  Boston  Med. 
and  Surg.  Jour.,  1913,  clxix,  741;  Kendall  and  Day,  ibid.,  1913,  clxix,  753. 


THE  PARATYPHOID  GROUP  351 

medium  (beta  type).  Isolation  from  the  feces  is  made  upon  Endo- 
plates  in  the  same  manner  that  dysentery  and  typhoid  bacilli  are 
obtained.  The  final  diagnosis  depends  upon  the  agglutination  of  the 
bacilli  with  specific  agglutinating  sera  of  high  potency.1 

(b)  Serological. — As  a  routine  measure  the  diagnosis  of  paratyphoid 
fever  by  the  agglutination  test  is  unreliable.  Not  infrequently  the 
blood  serum  of  a  patient  agglutinates  typhoid  bacilli  in  dilutions 
approaching  those  ultimate  for  the  homologous  organism.  The  para- 
typhoid bacilli  and  B.  typhosus  possess  in  common  group  agglutinins 
which  greatly  vitiate  the  value  of  the  test.  The  same  objection  does 
not  hold  for  the  diagnosis  of  typhoid  fever  by  the  agglutination 
reaction,  however. 

The  isolation  of  B.  paratyphosus  (alpha  or  beta)  from  the  blood 
stream  during  life,  or  from  the  internal  organs  at  autopsy  is  the  only 
reliable  method  of  diagnosis.  Carriers  are  not  uncommon,  and  like 
typhoid  bacillus  carriers  the  organisms  frequently  remain  in  the 
gall-bladder,  consequently  isolation  of  the  bacilli  from  feces  does  not 
necessarily  establish  a  correct  clinical  diagnosis.  Paratyphoid  bacilli 
have  been  isolated  occasionally  from  gall-stones  and  from  cases  of 
cholecystitis,  particularly  in  women. 

SUMMARY. 

THE  MORE  IMPORTANT  DIFFERENTIAL  DETAILS  OF  PARATYPHOID  FEVER  AND 
OF  MEAT  POISONING. 

Meat  Poisoning.  Paratyphoid  Fever. 

Organism      ....      Hog  cholera  bacillus.  B.  paratyphosus  alpha. 

B.  enteritidis.  B.  paratyphosus  beta. 

Habitat  of  organism      .      Intestinal  canal  of  lower  ani-  Chiefly  intestinal   tract  - 

mals  chiefly:  hog  cholera  of  man. 
in  swine,  enteriditis  com- 
mon in  rodents. 

Mode  of  infection     .      .      Usually  contaminated  meat  Usually  human   bacilli 

(human  carriers  rare).  carriers. 

Incubation  period     .      .      Six  to  forty-eight  hours.  Eight  to  twenty  days. 

Symptoms      ....      Choleraic.  Typhoidal. 

Pneumonic  Infection  with  B.  Psittacosis. — B.  psittacosis  causes  a 
fatal  enteritis  in  parrots,  and  it  has  been  noticed,  particularly  in 
France,  that  coincidently  with  enteric  disease  in  parrots  a  pneumonic 
infection  has  appeared  in  those  associated  with  them.  The  disease 
in  man  presents  no  definite  clinical  features  which  would  differentiate 
it  from  typhoid  fever  complicated  by  pneumonia.  Tke  incubation 

1  Sera  that  will  agglutinate  homologous  strains  in  dilutions  of  1  to  40,000  are  readily 
prepared;  such  sera  in  dilutions  of  1  to  10,000  may  be  regarded  as  specific  for  the  identi- 
fication of  members  of  the  group,  if  typical  agglutination  occurs. 


352  THE  ALCALIGENES-DYSENTERY— TYPHOID 

period  varies  from  five  days  to  three  weeks,  usually,  however,  less 
than  ten  days.  The  onset  is  gradual  in  some  cases,  like  typhoid,  but 
it  may  be  abrupt  with  an  initial  chill,  as  in  pneumonia.  The  spleen 
is  enlarged,  but  rose  spots  are  rarely  found.  The  mortality  varies; 
it  may  be  as  high  as  30  per  cent.  The  postmortem  lesions  have  not 
been  established.  In  one  case  the  bacillus  was  isolated  from  the  heart's 
blood  postmortem.  Specific  agglutinins  in  the  patient's  blood  serum 
have  not  been  satisfactorily  studied,  and  the  disease  as  a  clinical 
entity  is  yet  to  be  defined.  The  principal  evidence  of  the  causative 
relationship  of  B.  psittacosis  to  the  disease  rests  at  present  upon  the 
occasional  household  epidemics  following  closely  upon  the  presence  of 
a  diseased  parrot. 

Immunity  and  Immunization  to  Paratyphoid  Infection. — The  duration 
of  immunity  following  recovery  from  an  attack  of  paratyphoid  fever 
or  of  meat  poisoning  is  as  yet  undetermined.  The  brilliant  results 
of  protective  immunization  against  typhoid  fever  with  vaccines  or 
residues  of  the  typhoid  bacillus  have  led  to  similar  vaccination  against 
paratyphoid  infection  with  polyvalent  vaccines  composed  of  the 
principal  strains  of  the  paratyphoid  group.  Combined  protective 
vaccination  against  typhoid  and  paratyphoid  by  the  use  of  com- 
pound vaccines  has  also  been  attempted.  The  efficiency  of  the 
immunization  can  not  be  stated  at  the  present  time  because  statistics 
are  unavailable. 

Dissemination  and  Prophylaxis. — Paratyphoid  fever  appears  to  be 
spread  by  mild  unrecognized  cases,  by  carriers,  and  by  the  occasional 
transmission  of  bacilli  through  food,  water  or  milk.  Flies  may  also 
be  a  factor  in  the  dissemination  of  the  organisms.  Meat  poisoning 
is  chiefly  disseminated  by  infected  meats,  more  frequently  that  of 
cattle  or  swine.  The  customary  precautions  appropriate  for  excremen- 
titious  diseases,  including  the  restriction  of  carriers,  may  be  con- 
fidently relied  upon  to  prevent  the  spread  of  paratyphoid  fever. 
Thorough  cooking  will  largely  reduce  the  occasional  danger  from 
contaminated  meats. 


CHAPTER  XVII. 
THE  COLI— CLOACA— PROTEUS  GROUP. 

BACILLUS   COLL 

Historical. — Bacillus  coli  was  isolated  in  pure  culture  from  the 
feces  of  infants,  and  its  important  cultural  characters  determined  by 
Escherich  in  1886.1  It  is  very  probable,  as  Escherich  suggested,2 
that  Emmerich's  B.  neapolitanus,  Brieger's  "propionic  acid  bacillus," 
and  Frankel's  bacilli3  are  identical  with  the  colon  bacillus. 

Morphology. — Bacillus  coli  is  a  rod-shaped  organism  which  varies 
in  shape  from  oval  organisms  resembling  cocci  to  bacilli  of  moderate 
length.  The  organism  varies  in  size  from  0.5  to  0.8  micron  in  dia- 
meter and  from  1  to  3  microns  in  length.  The  bacilli  occur  singly  and 
in  pairs;  in  older  cultures  short  chains  and  elongated  organisms  are 
frequently  observed.  The  ends  are  distinctly  rounded.  Motility  is 
variable;  many  strains  are  non-motile  except  during  the  earlier  hours 
of  growth.  Young  cultures  on  gelatin  are  said  to  exhibit  motility 
when  older  growths  even  in  the  same  medium  are  motionless  except 
for  Brownian  movement.  Very  commonly  only  a  very  few  organisms 
in  a  microscopic  field  exhibit  motion,  the  remainder  being  without 
movement.  Four  to  eight  peritrichic  flagella  are  commonly  attached 
to  each  bacillus;  less  frequently  as  many  as  twelve  may  be  demon- 
strated. The  flagella  are  somewhat  shorter  than  those  of  the  typhoid 
bacillus  and  they  are  more  difficult  to  stain.  Bacillus  coli  forms  no 
spores  nor  capsules.  It  stains  readily  with  the  ordinary  anilin  dyes, 
ard  it  is  uniformly  Gram-negative. 

Isolation  and  Culture. — The  colon  bacillus  grows  readily  on  the  ordi- 
nary media;  the  superficial  colonies  on  agar  plates  are  clear  and  color- 
less and  attain  a  diameter  of  from  2  to  5  mm.  after  ei»iteen  hours' 
incubation  at  379  C.  If  the  surface  of  the  medium  is  mowt  the  edges 
of  the  colonies  are  somewhat  irregular  in  outline;  on  dry  surfaces  the 
colonies  are  round  and  slightly  convex  in  section.  Viewed  by  trans- 

1  Die  Darmbakterien  des  Sauglings,  Stuttgart,  1886,  63J 

2  Loc.  cit.,  73,  74. 

3  Deutsch.  med.  Wchnschr.,  1885,  Nos.  34  and  35. 
23 


354  THE  COL/— CLOACA— PROTEUS  GROUP 

mitted  light  the  growths  are  yellowish-brown;  by  reflected  light  they 
are  colorless.  Colonies  on  gelatin  develop  more  slowly  and  become 
somewhat  brownish  in  color.  The  medium  is  not  liquefied.  Rapid 
development  occurs  in  plain  and  sugar  broths.  A  heavy,  brownish 
spreading  growth  occurs  on  the  surface  of  slanted  potato. 

Bacillus  coli  is  an  aerobic,  facultatively  anaerobic  organism  which 
grows  best  at  37°  C.  Growth  ceases  below  8°  to  10°  C.,  and  above 
43°  to  45°  C.  An  exposure  of  fifteen  minutes  at  75°  C.  kills  them.  In 
general  the  colon  bacillus  is  somewhat  more  resistant  to  physical  and 
chemical  agents  than  the  typhoid  bacillus. 

Products  of  Growth. — (a)  Chemical. — Bacillus  coli  produces  indol 
from  tryptophan  in  sugar-free  media,  and  phenolic  bodies  from 


FIG.  48.— Bacillus  coli  flagella.      X  1500.      (Kplle  and  Hetsch.) 

tyrosine  under  the  same  conditions.  Hydrogen  sulphide  and  ammonia, 
the  latter  resulting  largely  from  deaminization  of  proteins  and 
protein  derivatives,  are  also  produced  in  considerable  amounts  in 
media  containing  no  utilizable  carbohydrates.1  Similar  products 
may  be  formed  in  the  intestinal  tract  under  certain  conditions.  The 
addition  of  utilizable  carbohydrates  to  protein  media  changes  the 
character  of  the  products  of  metabolism  in  a  noteworthy  manner. 
Under  these  conditions  the  protein  constituents  of  the  media  are 
practically  unchanged;  the  sugars  are  fermented  with  the  production 
of  carbon  dioxide  and  hydrogen,2  lactic  acid  and  smaller  amounts  of 

1  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Soc.,  1913,  xxxv,  1228. 

2  In  the   proportion  H :  CO2  =  f .     Theobald  Smith,  The  Fermentation  Tube.     The 
Wilder  Quarter  Century  Book,  1893,  p.  202.     Very  exact  determinations  of  the  gaseous 
products  of  fermentation  of  B.  coli  have  been  made  by  Harden  and  Walpole,  Proc.  Roy. 
Soc.,  1906,  77,  399. 


BACILLUS  CO  LI  355 

acetic  acid  and  formic  acid.  Dextrose,  lactose  and  mannite  are  thus 
fermented;  saccharose  is  not  decomposed  by  the  strains  of  the  colon 
bacillus  commonly  found  in  the  intestinal  tract.  Occasionally  a  sac- 
charose-fermenting strain  is  encountered  in  the  feces.1 

The  reactions  of  the  colon  bacillus  in  milk  are  variable;  typical 
strains  produce  enough  acid  from  the  fermentation  of  the  lactose  to 
cause  an  acid  coagulation  in  one  to  three  days  at  37°  C.  Neutraliza- 
tion of  the  acid  by  alkali  redissolves  the  coagulum  and  the  medium 
resumes  its  normal  appearance.  Occasional  strains  do  not  cause 
coagulation  even  after  boiling  the  milk.2  Gas  is  not  produced  in 
appreciable  amounts  in  milk  by  B.  coli,  and  the  organism  leaves  the 
milk  proteins  practically  intact  even  after  prolonged  incubation — 


FIG.  49. — Bacillus  coli,  broth  culture. 

the  carbohydrate  constituents  alone  'are  acted  upon.3  Coagulation 
does  not  as  a  general  rule  occur  in  litmus  milk,  but  boiling  the  medium 
usually  causes  rapid  clotting.  The  ordinary  litmus  of  commerce 
contains  considerable  amounts  of  calcium  carbonate.  This  may 
neutralize  seme  of  the  acid  products  of  fermentation,  reducing  the 
acidity  below  the  coagulation  point.  This  explanation  does  not 
account  for  the  same  phenomenon  in  milk  colored  with  pure  litmus 
or  azolitmin.  Gelatin  is  not  liquefied  by  B.  coli.  Nitrates  are  reduced 
to  nitrites. 

(6)  Enzymes. — Soluble  proteolytic  and  lipolytic  enzymes  have  not 

been  detected  in  cultures  of  Bacillus  coli.    Buxton4  has  demonstrated 

• 

1  Theobald  Smith,  Am.  Jour.  Med.  ScM  September,  1895. 

2  Ibid.,  Fermentation  Tube,  p.  201. 

3  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Soc.,  1914,  xxxvii,  1945, 
<Am.  Med.,  1903,  vi,  137. 


356  THE  COLI— CLOACAE— PROTEUS  GROUP 

both  a  maltase  and  a  lactase  in  maltose  and  lactose  cultures  of  the 
organism  respectively.  The  investigations  of  Franzen  and  Stuppuhn1 
would  suggest  that  the  liberation  of  gas  in  sugar  broth  cultures  of 
B.  coli  and  other  aerogenic  bacteria  depends  upon  the  production 
of  formic  acid  from  the  carbohydrate  and  its  subsequent  decomposi- 
tion into  carbon  dioxide  and  hydrogen  by  the  action  of  an  enzyme, 
formiase,  in  accordance  with  the  equation  H.COOH  =  CO2  +  H2. 

(c)  Toxins. — Bacillus  coli  does  not  produce  a  soluble  toxin.  The 
injection  of  killej  cultures  into  laboratory  animals  frequently  causes 
death;  if  large  amounts  are  introduced  intravenously  into  rabbits 
there  is  usuaUy  a  lowering  of  the  body  temperature,  diarrhea,  collapse 
and  death  even  within  three  hours.2  If  the  animals  survive  for  a 
longer  time  a  purulent  peritonitis  may  develop.  Living  cultures  of 
colon  bacilli  derived  from  inflammatory  processes  in  man  are  gen- 
erally virulent  for  guinea-pigs.  Old  stock  cultures  ark  less  virulent 
as  a  rule.  The  symptoms  of  toxemia  which  are  exhibited  by  labor- 
atory animals  following  the  injection  of  colon  bacilli  are  probably 
caused  by  the  liberation  of  endotoxins  from  the  bacilli. 

Pathogenesis. — The  colon  bacillus  is  a  normal  inhabitant  of  the 
intestinal  tracts  of  man  and  the  higher  animals.  Ordinarily  it  is  a 
harmless  parasite,  but  it  may  become  invasive  if  conditions  arise 
which  weaken  the  intestinal  mucosa.  In  peritonitis,  purulent  per- 
forative  appendicitis,  angiocholitis,  and  even  in  occasional  cases  of 
pancreatitis  the  organism  is  frequently  isolated,  either  in  pure  culture 
or  in  association  with  other  bacteria,  as  streptococci,  typhoid  bacilli, 
or  staphylococci.  It  is  difficult  to  determine  with  precision  the  part 
played' by  Bacillus  coli  in  these  conditions.  Occasional  cases  of  enter- 
itis are  encountered  which  appear  to  be  caused  by  this  organism, 
other  bacteria  having  been  ruled  out.  The  careful  studies  of  Coleman 
and  Hastings3  are  of  great  importance  in  this  connection.  They 
isolated  colon  bacilli  from  the  blood  stream  in  a  small  series  of  cases 
which  presented  symptoms  indistinguishable  from  those  of  typhoid 
fever.  No  typhoid  bacilli  were  ever  found  in  these  patients,  and  no 
specific  agglutinins  for  the  typhoid  bacillus  were  demonstrable. 
Specific  agglutinins  for  the  homologous  strains  of  B.  coli  persisted 
until  recovery.  Cystitis  and  pyelonephritis,  particularly  the  former, 
are  frequently  found  to  be  a  pure  colon  infection.  B.  coli  is  occa- 

1  Ztschr.  f.  physiol.  Chem.,  1912,  Ixxvii,  129. 

2  Escherich,  Fort.  d.  Med.,  1885,  521. 

3  Med.  and  Surg.  Report  of  Bellevue  and  Allied  Hospitals  of  the  City  of  New  York, 
1909-1910,  iv,  56. 


BACILLUS  COLI  357 

sionally  isolated  from  the  centre  of  gall-stones;  it  is  surmised  that 
the  organism,  or  clusters  of  them,  act  as  nuclei  around  which  the 
cholesterin  is  gradually  deposited.  Colon  bacilli  have  been  isolated 
in  rare  instances  from  purulent  cerebrospinal  fluids,  and  they  may 
cause  bronchopneumonia.  Perirectal  abscesses  also  may  contain  pure 
cultures  of  colon  bacilli. 

Immunity  and  Immunization. — The  constant  occurrence  of  B.  coli  in 
large  numbers  in  the  normal  intestinal  tract  is  an  index  of  the  rela- 
tive immunity  of  man  to  infection  with  this  organiaji.  Occasionally 
very  small  numbers  of  bacilli  may  gain  entrance  to  the  tissues,  par- 
ticularly in  young  children.  The  blood  serum  usually  contains  agglu- 
tinins  in  small  amounts  for  the  organism.  In  practice  no  attempt  is 
made  to  increase  the  immunity  to  colon  bacilli,  except  in  cases  of 
cystitis  or  other  local  infection.  Vaccines  of  the  homologous  strain 
of  B.  coli  are  occasionally  administered  in  such  instances.  The  results 
have  been  variously  interpreted. 

Bacteriological  Diagnosis. — The  methods  of  isolation,  identification 
and  significance  of  B.  coli  in  water  supplies  will  be  discussed  in  the 
chapter  on  water.  Isolation  of  colon  bacilli  from  the  intestinal  con- 
tents or  feces  is  readily  accomplished  by  plating  methods.  The 
organisms  far  outnumber  any  others  normally  present,  and  even  in 
severe  diarrheal  disorders  colon  bacilli  do  not  entirely  disappear. 
Prolonged  starvation  does  not  eliminate  B.  coli  from  the  intestinal 
canal.1  The  morphology  and  staining  reactions  are  not  distinctive. 
Plating  methods — principle  involved:  lactose  agar,  containing  lit- 
mus or  decolorized  fuchsin  (Endo  medium)  as  an  indicator  is  infected 
with  material  suspected  to  contain  B.  coli.  The  organism  ferments 
the  lactose  with  the  production  of  acid;  the  acid  changes  the  color 
of  the  indicator  immediately  surrounding  the  colon  bacilli,  red  if 
litmus  is  used,  pink  if  fuchsin  is  employed.  The  red  colonies  are 
inoculated  into  broth  and  incubated  to  obtain  sufficient  organisms 
for  their  identification  by  cultural  methods. 

Cultural  Identification. — A  Gram-negative  bacillus  which  produces 
gas  in  dextrose,  lactose  and  mannite  (optionally  in  saccharose),  coagu- 
lates but  does  not  'peptonize  milk,  does  not  liquefy  gelatin,  and  is 
without  action  upon  starches  is  Bacillus  coli. 

1  At  the  end  of  thirty-one  days'  abstinence  from  all  food,  typical  colon  bacilli  were 
present  in  the  lower  part  of  the  large  intestine.  Kendall,  Observations  upon  the  Bacterial 
Intestinal  Flora  of  a  Starving  Man,  Publication  No.  203  of  the  Carnegie  Institute  of 
Washington,  1915,  p.  232.  This  experiment  emphasizes  the  fallacy  of  "starving  out" 
intestinal  bacteria  by  withdrawing  food. 


358  THE  COLI— CLOACA— PROTEUS  GROUP 

BACILLUS   CLOAOffi. 

Bacillus  cloacae  was  isolated  from  sewage  and  polluted  water  by 
Jordan.1  The  organism  appears  to  be  relatively  abundant  some  years 
and  comparatively  uncommon  other  years.  When  it  is  abundant  in 
sewage  it  is  found  occasionally  in  the  intestinal  tract  of  man. 

Morphology. — The  bacillus  is  of  moderate  size,  measuring  from  0.6 
to  0.8  micron  in  diameter  and  from  1  to  2  microns  in  length.  It 
occurs  singly  or  in  pairs,  uncommonly  in  short  chains.  Young  cultures 
exhibit  motility,  and  the  organisms  possess  peritrichic  flagella.  No 
spores  or  capsules  have  been  demonstrated.  Ordinary  anilin  dyes 
color  the  bacilli  readily,  and  they  are  Gram-negative. 

Isolation  and  Culture. — The  colonies  on  agar  plates  after  eighteen 
hours'  incubation  are  round,  clear  and  colorless,  and  measure  from 
1  to  3  mm.  in  diameter.  There  is  nothing  distinctive  in  the  appear- 
ance of  the  growths. 

Products  of  Growth. — (a)  Chemical. — Indol,  phenol,  hydrogen  sul- 
phide and  ammonia  are  produced  in  sugar-free  broth.  The  ammonia 
production  is  greater  than  that  characteristic  of  B.  coli  and  less  than 
that  ordinarily  produced  by  B.  proteus.2  Acid  and  gas  are  produced 
in  dextrose,  lactose,  saccharose  and  mannite  broths.  The  gas  ratio 
is  somewhat  variable,  but  distinctive ;  the  proportion  of  carbon  dioxide 
to  hydrogen  is  greater  than  that  produced  by  other  closely-related 
bacteria.3  The  action  of  the  organism  upon  lactose  is  slow,  and  less 
gas  is  produced  from  this  sugar.  The  amount  of  gas  produced  from 
dextrose  and  saccharose  is  greater  than  that  produced  by  other  aero- 
genie  members  of  the  paratyphoid-pro teus  group.  B.  cloacae  forms 
but  little  acid  from  the  fermentation  of  sugars,  and  after  one  to  three 
days  the  reaction,  even  in  sugar  broth,  becomes  alkaline,  due  to  the 
exhaustion  of  the  sugar  and  the  subsequent  decomposition  of  the 
protein  constituents  of  the  broth.4  Indol  and  other  products  of  putre- 
faction are  formed  as  soon  as  the  sugar  is  exhausted. 

Milk  is  coagulated  and  slowly  peptonized.  Freshly  isolated  cultures 
usually  liquefy  gelatin,  but  this  property  is  lost  after  prolonged  artifi- 
cial cultivation. 

The  organism  is  ordinarily  non-pathogenic  for  man. 


1  Annual  Report  of  Massachusetts  State  Board  of  Health,  1890,  p.  836. 

2  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Soc.,  1913,  xxxv,  1230. 
3H:CO2  =  £  -  i     Theobald  Smith,  Fermentation  Tube,  1893,  p.  215. 
4  Kendall,  Day  and  Walker,  loc.  cit. 


BACILLUS  PROTEUS  GROUP  359 

BACILLUS   PROTEUS    GROUP. 

Synonyms. — Proteus  vulgaris,  Proteus  mirabilis,  Proteus  Zenkeri, 
Proteus  Zopfii,  Proteus  fluorescens. 

Historical. — The  proteus  group  comprises  several  closely-related 
bacilli  found  commonly  in  soil,  in  water  rich  in  organic  matter,  as 
sewage,  in  human  feces,  and  associated  with  the  decay  of  organic 
matter.  The  important  members  of  the  group  were  first  isolated 
in  pure  culture  and  described  by  Hauser.1 

Morphology. — The  proteus  bacilli  are  rod-shaped  organisms  of  vari- 
able length  which  occur  singly  and  in  pairs  as  a  rule;  less  commonly 
they  remain  adherent  in  short  chains.  The  size  of  individual  cells 
varies  considerably,  even  in  the  same  culture.  The  limits  of  varia- 
tion are  comprised  within  the  following  dimensions:  diameter  from 
0.6  to  0.8  micron,  length  from  1.0  to  3.5  microns.  Proteus  bacilli 
are  actively  motile  and  possess  a  large  number  of  peritrichic  flagella2 
which  are  frequently  seen  as  a  tangled  filamentous  mass  surrounding 
each  individual  cell.3  Special  staining  methods  are  required  for  the 
demonstration  of  these  flagella.  The  organisms  produce  no  spores 
and  form  no  capsules.  They  stain  with  ordinary  anilin  dyes,  but 
somewhat  faintly,  and  they  are  Gram-negative. 

Isolation  and  Culture. — The  members  of  the  proteus  group  develop 
rapidly  on  gelatin  at  room  temperature;  the  organisms  typically 
liquefy  the  medium  with  great  rapidity.  Some  strains  liquefy  gelatin 
but  slightly  or  even  not  at  all.  The  colonies  of  rapidly  liquefying 
strains  in  5  per  cent,  gelatin  are^  frequently  very  characteristic;  the 
organisms  tend  to  remain  adherent,  forming  masses  of  bacilli  which 
slowly  move  around  in  an  area  of  liquefied  gelatin.  Hauser4  recognized 
four  types  of  proteus  bacilli  classified  according  to  their  ability  to 
liquefy  gelatin:  Proteus  vulgaris  liquefies  gelatin  rapidly;  Proteus 
mirabilis  liquefies  gelatin  slowly;  Proteus  zenkeri  and  Proteus  zopfii 
do  not  liquefy  this  medium.  The  latter,  Proteus  zopfii,  exhibits 
negative  geotropism  on  slanted  solid  media.  It  is  now  recognized 
that  cultures  of  B.  proteus  may  gradually  lose  their  gelatin-liquefying 
power  after  prolonged  cultivation,  so  that  a  cultural  transition  from 
B.  proteus  to  B.  zenkeri  may  be  observed  in  the  laboratory.  A  dis- 

1  Ueber  Faulnisbakterien  und  deren  Beziehungen  zur  Septikamie,  Leipzig,  1885. 

2  Zettnow,  Centralbl.  f.  Bakt.,  1891,  x,  689. 

3  Massea  (Centralbl.  f.  Bakt.,  1891,  ix,   106)  states  that  young  bacilli  may  possess 
from  60  to  100  flagella. 

4  Loc.  cit. 


360 


THE  COL/— CLOACA— PROTEUS  GROUP 


tinction  between  the  three  types  is  no  longer  made.    It  is  not  deter- 
mined whether  B.  zopfii  is  a  separate  variety  of  B.  proteus. 

The  organisms  grow  vigorously  in  milk,  causing  slight  acidification 
and  peptonization.  The  development  in  broth  is  equally  vigorous; 
acid  and  gas  are  produced  in  dextrose  and  saccharose  broths.1  Neither 
acid  nor  gas  is  formed  in  lactose  broth.2 

Proteus  bacilli  grow  slowly  at  0°  C.3  and  at  temperatures  not 
exceeding  43°  to  45°  C.  The  optimum  temperature  is  about  25°  C. 
but  development  is  rapid  at  37°  C.  Strains  obtained  from  putrefying 
organic  matter  are  tolerant  of  considerable  degrees  of  alkalinity4 
and  acidity;5  those  from  the  human  body  are  somewhat  less  tolerant. 
The  growth  of  B.  proteus  at  low  temperatures  is  of  considerable  prac- 


FIG.  50. — Bacillus  proteus,  flagella  stain.      X  1500.     (Gunther.) 


tical  importance;  several  cases  of  ptomain  poisoning  have  been 
attributed  to  foods  decomposed  by  this  organism  at  the  temperature 
of  the  ice-box.  The  resistance  of  the  organisms  to  heat  is  not  great. 
According  to  Meyerhof,6  an  exposure  of  twenty-five  to  thirty-five 
minutes  at  54°  C.,  five  to  ten  minutes  at  56°  C.,  and  of  one-half  a 
minute  at  60°  C.,  kills  them.  Their  resistance  to  disinfectants  is 
similar  to  that  of  B.  coli. 

Products  of  Growth. — (a)  Chemical.— Proteus  bacilli  decompose 
proteins  and  protein  derivatives  energetically.  The  following  sub- 
stances have  been  detected  among  the  cleavage  products:  trimethy- 
lamine,  betain,  phenol,  hydrogen  sulphide  ;7  from  the  decomposition  of 

1  Theobald  Smith,  Fermentation  Tube,  Wilder  Quarter  Century  Book,  1893,  p.  213. 

2  The  bacilli  may  gradually  lose  their  ability  to  ferment   saccharose;   strains  which 
do  not  ferment  this  sugar  may  be  mistaken  for  paratyphoid  bacilli,  particularly  if  the 
gelatin-liquefying  power  disappears  simultaneously.     The  very  considerable  production 
of  ammonia  in  sugar-free  broth  readily  distinguishes  the  proteus  bacilli.     Kendall,  Day, 
and  Walker,  Jour.  Am.  Chem.  Soc.,  1913,  xxxv,  1231. 

3  Levy,  Arch.  f.  offentl.  Gesundhpf.  in  Els.  Lothr.,  1895,  xvi,  Heft  3. 

4  Deelman,  Arb.  a.  d.  kais.  Gesamte,  1897,  xiii,  374. 
6  Fermi,  Centralbl.  f.  Bakt.,  1898,  xxiii,  208. 

6  Centralbl.  f.  Bakt,,  1898,  xxiv,  20. 
7Emmerling,  Ber.  chem.  Gesell.,  1896,  2711. 


BACILLUS  PROTEUS  GROUP  361 

casein,  deuteroalbumose,  peptone,  mono-  and  diamino-acids  (histidin 
and  lysin),  tyrosin,  indol,  and  skatol.1  An  extensive  liberation  of 
ammonia  takes  place  in  protein  media  free  from  sugars.2  Ammonia 
is  also  formed  from  the  proteins  of  milk,  but  more  slowly,  and  in 
smaller  amounts.3  Carbon  dioxide  and  hydrogen  (H :  CO2  —  f)  are 
formed  in  dextrose  and  saccharose  broths,  together  with  lactic  acid 
and  small  amounts  of  formic  acid.  Lactose  is  unfermented.4  Urea 
is  actively  decomposed,  ammonia  and  carbon  dioxide  being  liberated.5 
The  addition  of  dextrose  prevents  the  liberation  of  ammonia  and 
carbon  dioxide.6 

(b)  Enzymes. — B.  proteus  produces  a  soluble  proteolytic  enzvme 
in  protein  media  containing  no  utilizable  sugars,  which  liquefies  egg 
albumen,  fibrin,  blood  serum,  and  gelatin.    This  enzyme  is  not  pro- 
duced when  utilizable  sugars  are  present  in  the  medium.     No  other 
enzymes  are  known. 

(c)  Toxins. — A  soluble  toxin  has  not  been  demonstrated  in  cultures 
of  B.  proteus.    At  one  time  "sepsin"  (see  page  75)  was  supposed  to 
be  an  important  factor  in  "ptomain  poisoning."    This  substance  is 
produced  in  but  minute  amounts  by  proteus  bacilli,  however,  and 
no  importance  is  attached  to  it.    The  nature  of  the  poisonous  substance 
produced  by  B.  proteus  is  unknown. 

Pathogenesis. — Several  types  of  disease  have  been  attributed  to 
members  of  the  proteus  group.  Meat  poisoning  and  ptomain  poison- 
ing epidemics  caused  by  eating  meats  decomposed  by  the  organisms 
have  been  reported  by  Levy,7  Wesenberg,8  Silberschmidt,9  and  Pfuhl.10 
Dieudonne11  has  described  an  epidemic  which  originated  in  a  potato 
salad  from  which  proteus  bacilli  were  isolated.  B.  proteus  is  one  of 
the  very  few  bacteria  which  will  cause  cystitis  when  it  is  injected 
into  the  urinary  bladder.  Cystitis  in  man  is  frequently  caused  by 
B.  proteus.12  Pyelonephritis,  frequently  of  a  very  purulent  type,  and 
abscesses  are  occasionally  caused  by  members  of  the  group.  The 
organisms  do  not  as  a  rule  grow  in  normal  tissues,  but  they  grow 

1  Taylor,  Ztschr.  f.  physiol.  Chem.,  1902,  xxxvi. 

2  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Soc.,  1913,  xxxv,  1232^ 

3  Ibid.,  1914,  xxxvi,  1945. 

4  Theobald  Smith,  Fermentation  Tube,  Wilder  Quarter  Century  Book,  1893,  p.  213. 

5  Schnitzler,  Centralbl.  f.  Bakt.,  1893,  xiv,  219. 

6  Brodmeier,  Centralbl.  f.  Bakt.,  1895,  xviii,  380. 

7  Arch.  f.  exp.  Path.  u.  Pharm.,  1895,  xxxiv,  342. 

8  Ztschr.  f.  Hyg.,  1898,  xxviii,  484. 

9  Ibid.,  1899,  xxx,  328. 

10  Ibid.,  1900,  xxxv,  265. 

11  Miinchen.  med.  Wchnschr.,  1903,  2282. 

12  See  Meyerhof.  Centralbl.  f.  Bakt.,  1898,  xxiv,  18,  55,  148. 


362  THE  COL/— CLOACA— PROTEUS  GROUP 

readily  in  necrotic  tissues,  forming  much  pus  which  has  a  foetid  odor. 
Middle  ear  infections,  characterized  by  very  foul-smelling  pus,  have 
been  reported. 

Bacillus  proteus  fluorescens,  an  organism  exhibiting  many  charac- 
teristics of  the  proteus  group,  has  been  isolated  from  several  cases  of 
Weil's  disease  (infectious  jaundice)  by  Jaeger,1  Conradi  and  Vogt,2 
and  Bruning.3  Bar  and  Renon4  isolated  a  similar  bacillus  from  a  case 
of  jaundice  in  the  newborn.  Booker5  has  isolated  B.  proteus  from 
the  feces  of  a  large  number  of  cases  of  acute  summer  diarrhea  in 
children.  It  would  appear  from  his  studies  that  the  organisms  played 
a  prominent  part  in  the  causation  of  certain  types  of  this  illness,  par- 
ticularly those  characterized  by  choleraic  symptoms. 

Bacillus  proteus  is  not  very  pathogenic  for  laboratory  animals. 
The  injection  of  large  doses  usually  causes  death. 

Bacteriological  Diagnosis. — Bacillus  proteus  is  readily  isolated  upon 
gelatin  plates:  the  bacilli  grow  rapidly  at  room  temperature  and 
liquefy  the  medium  around  each  individual  colony.  Subcultures  in 
sugar  media,  gelatin  and  milk  produce  the  changes  outlined  above. 
B.  proteus  mav  be  confused  with  B.  cloacae,  because  the  latter  organ- 
ism ferments  lactose  more  slowly  than  other  sugars.6  B.  cloacae,  how- 
ever, is  distinctly  less  proteolytic  than  B.  proteus,7  and  it  produces 
less  acid  and  more  gas  from  dextrose. 

1  Zeit.  f.  Hyg.,  1892,  xii.  2  Ibid.,  1901,  xxxvii,  283. 

3  Deut.  med.  Woch.,  1904,  1269.  4  Sem.  med.,  1895,  234. 

5  Johns  Hopkins  Hospital  Reports,  vi. 

6  Theobald  Smith,  Fermentation  Tube,  1893,  215. 

7  Kendall,  Day  and  Walker,  loc.  cit.,  1230. 


CHAPTER  XVIII. 


THE  MUCOSUS  CAPSULATUS  GROUP. 


THE  Mucosus  CAPSULATUS  GROUP. 
Bacillus  Rhinoscleromatis. 


Bacillus  Ozsense. 
Bacillus  Lactis  Aerogenes. 


THE  first  member  of  the  bacteria  commonly  known  as  the  pneumo- 
Bacillus  Group  or  the  Mucosus  Capsulatus  Group  was  isolated  by 
Friedlander1  from  pneumonic  lungs.  At  that  time  he  believed  his 
"pneumonia  micrococcus"  was  the  causative  agent  of  lobar  pneu- 
monia, and  it  was  so  regarded  until  Frankel2  and  Weichselbaum3 
pointed  out  its  comparative  infrequency  in  lobar  pneumonia,  and 
differentiated  it  clearly  from  the  pneumococcus,  the  true  etiological 
organism  of  this  disease.  Weichselbaum  also  correctly  interpreted 
its  morphology  and  conferred  upon  it  the  name  Bacillus  pneumonise. 
Subsequent  investigations  by  many  observers  have  added  several 
closely-related  bacteria  to  the  group  which  at  the  present  time  com- 
prises the  following  somewhat  imperfectly-differentiated  types: 
Bacillus  mucosus  capsulatus  (Friedlander's  pneumobacillus),  Bacillus 
rhinoscleromatis,4  Bacillus  ozfense,5  Bacillus  lactis  aerogenes,6  and 
Bacillus  acidi  lactici.7 

Morphology. — The  members  of  the  Mucosus  Capsulatus  Group  are 
bacilli  which  vary  in  size  and  shape  in  the  same  culture  from  oval 
almost  coccoid  elements  to  distinctly  elongated  rods.  The  limits  of 
size  are  comprised  practically  within  the  following  dimensions :  diam- 
eter, 0.5  to  1.5  microns,  length,  0.6  to  3.5  microns.  They  occur 
typically  singly  or  in  pairs,  less  commonly  united  in  short  chains. 
Motility  is  not  observed  in  cultures  of  any  members  of  the  group  and 
they  appear  to  be  devoid  of  flagella.  Spores  have  not  been  detected. 
A  well-defined  capsule,  readily  demonstrable  by  capsule  stains,  sur- 
rounds each  organism  if  it  is  examined  in  tissues  or  secretions  of  the 
animal  body,  or  in  albuminous  media.  It  tends  to  disappear  during 

1  Virchows  Arch.,  1882,  Ixxxvii,  319;  Fort.  d.  Med.,  1883,  i,  719. 

2  Ztschr.  f.  klin.  Med.,  1886,  x,  401. 

3  Wien.  med.  Jahrb.,  1886. 

*  V.  Frisch,  Wien.  med.  Wchnschr.,  1882,  No.  32. 

8  Abel,  Ztschr.  f.  Hyg.,  1896,  xxi,  89;    Centralbl.  f.  Bakt.,  1893,  xiii,  161. 

6  Escherich,  Darmbakterien  des  Sauglings,  Stuttgart,  1886,  p.  57. 

7  Hueppe,  Deutsch.  med.  Wchnschr.,  1884,  p.  778. 


364  THE  MUCOSUS  CAPSULATUS  GROUP 

prolonged  cultivation  in  the  usual  artificial  laboratory  media.  Ordi- 
nary anilin  dyes  color  the  organisms  readily,  and  they  are  Gram- 
negative. 

Isolation  and  Culture. — The  members  of  the  Mucosus  Capsulatus 
Group  grow  readily  on  artificial  media.  The  colonies  on  agar  are  white 
or  gray,  from  1.5  to  3  mm.  in  diameter,  very  viscid,  and  raised;  they 
tend  to  become  confluent.  When  touched  with  a  platinum  needle 
the  growth  may  be  drawn  away  as  a  tenacious,  sticky  filament.  In 
gelatin,  a  non-characteristic  filamentous  growth  occurs  along  the  line 
of  inoculation  and  the  surface  becomes  covered  with  a  white,  glisten- 
ing raised  colony.  The  gelatin  is  not  liquefied.  Milk  is  acidified,  and 


FIG.  51. — Bacillus  mucosus  capsulatus.      X  1000. 

frequently  the  accumulation  of  acid  leads  to  coagulation.  A  light- 
pink  color  is  imparted  to  litmus  milk  and  coagulation  is  irregular  in 
this  medium.  Broth  is  clouded,  and  a  slimy,  viscid  sediment  collects 
at  the  bottom  of  the  tube.  A  majority  of  strains  produce  gas  bubbles 
on  potato. 

The  organisms  are  aerobic,  facultatively  anaerobic.  Growth  takes 
place  at  8°  to  10°  C.,  but  37°  C.  is  the  optimum  temperature.  Little 
or  no  growth  occurs  above  43°  C. 

Products  of  Growth. — The  majority  of  strains  do  not  form  indol, 
but  occasional  cultures  give  a  marked  reaction  for  this  substance.1 
Practically  all  strains  form  a  mucinous  substance  on  artificial  media. 

The  reactions  of  fermentation  have  been  used  as  a  basis  for  separa- 
tion into  types  by  Perkins,2  who  groups  the  organisms  in  the  following 
manner : 

1  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Soc.,  1913,  xxxv,  1237. 

2  Jour.  Infec.  Dis.,  1904,  i,  241. 


BACILLUS  OZMNM  365 


Type  I.  —  All  carbohydrates  and  starch  fermented  with  the  produc- 
tion of  gas  (H:CO2  =  r)  and  acid;  Bacillus  lactis  aerogenes. 

Type  II.  —  All  carbohydrates  except  saccharose  fermented;  starch 
fermented  —  Bacillus  mucosus  capsulatus,  Bacillus  rhinoscleromatis, 
Bacillus  ozsenae. 

Type  III.  —  All  carbohydrates  except  saccharose  and  starch  fer- 
mented; Bacillus  acidi  lactici. 

Enzymes  and  toxins  have  not  been  demonstrated  in  cultures  of  any 
members  of  the  group. 

Pathogenicity.  —  Human.  —  Bacillus  mucosus  capsulatus  has  been 
isolated  in  a  considerable  proportion  of  cases  of  lobular  pneumonia, 
but  it  practically  never  is  the  sole  incitant  of  lobar  pneumonia.  It 
is  occasionally  detected  in  purulent  inflammations  of  the  respiratory 
tract  not  pneumonic  in  character,  in  the  purulent  secretions  of  the 
nasal  and  frontal  sinuses,  in  occasional  cases  of  pericarditis  and 
pleurisy,  stomatitis  and  otitis  media.  The  normal  sputum  occasionally 
contains  the  organism. 

Animal.  —  Subcutaneous  inoculations  into  mice,  rabbits  or  guinea- 
pigs  frequently  lead  to  abscess  formation  characterized  by  thick, 
viscid  pus.  Occasionally  a  generalized  infection  which  results  fatally 
takes  place. 

Bacillus  Rhinoscleromatis.  —  Rhinoscleroma,  characterized  by 
indurated  granulomatous  nodules  of  the  mucous  membrane  of  the 
nose,  is  ascribed  to  Bacillus  rhinoscleromatis  by  v.  -Frisch,1  Paltauf 
and  v.  Eiselsberg,2  and  others.  A  satisfactory  demonstration  of  the 
etiology  of  this  infection  is  wanting,  but  organisms  culturally  like 
Bacillus  rhinoscleromatis  have  been  isolated  from  the  cells  of  Miculicz, 
large,  swollen  cells  with  crescentric  nuclei  characteristically  present  in 
rhinoscleroma  and  demonstrated  within  them  on  section. 

Bacillus  Ozsense.  —  Ozena,  a  disease  of  the  nose  characterized  by  a 
fetid  catarrhal  inflammation,  is  very  frequently  associated  with  the 
presence  of  large  numbers  of  a  member  of  the  Mucosus  Capsulatus 
Group  to  which  Abel3  gave  the  name  Bacillus  ozsense.  The  organism 
has  not  been  sharply  separated  from  Bacillus  rhinoscleromatis  and 
Bacillus  mucosus  capsulatus,  and  its  etiological  relationship  to  ozena 
is  still  sub  judice.  Autogenous  vaccines  of  the  organism  have  been 
used  with  varying  success  in  the  treatment  of  the  disease. 

1  Loc.  cit. 

2  Fort.  d.  Med.,  1886,  Nos.  19  and  20. 

3  Loc.  cit. 


366  THE  MUCOSUS  CAPSULATUS  GROUP 

Bacillus  Lactis  Aerogenes. — This  organism  is  an  almost  constant 
inhabitant  of  the  upper  part  of  the  intestinal  tract  of  nurslings;  it  is 
common  in  the  intestinal  contents  of  bottle-fed  infants,  and  it  fre- 
quently persists  in  small  numbers  in  the  adult  intestinal  tract.  A 
closely  related  organism,  Bacillus  acidi  lactici,  is  found  fairly  widely 
distributed  in  milk,  water,  and  sewage.  A  sharp  differentiation  between 
the  two  organisms  is  difficult  to  establish.  There  is  evidence  that 
the  organism,  ordinarily  a  harmless  intestinal  parasite,  may  become 
temporarily  pathogenic  and  incite  intestinal  disturbance  varying  in 
intensity  from  slight  diarrhea  to  severe  enteritis.1  Occasional  cases 
of  cystitis  in  infants  are  also  associated  with  the  presence  of  Bacillus 
lactis  aerogenes  in  pure  culture. 

It  is  obvious  that  the  interrelations  of  the  Mucosus  Capsulatus  Group 
are  at  present  in  an  unsatisfactory  state — attempts  to  separate  the 
organisms  on  the  basis  of  serological  reactions  have  been  unsuccessful, 
partly  because  of  the  difficulty  of  removing  the  capsules  which  appear 
to  be  somewhat  impervious  to  antibodies.  A  final  arrangement  of 
the  group  and  an  ultimate  differentiation  of  the  various  organisms 
comprising  it  awaits  future  elucidation. 

1  Kendall  and  Day,  Boston  Med.  and  Surg.  Jour.,  1913,  clxix,  753;  Kendall,  ibid., 
May  20,  1915. 


CHAPTER  XIX. 

GLANDERS,  ANTHRAX,  PYOCYANEUS,  INFECTIOUS 
ABORTION:    ACIDURIC  BACTERIA. 

BACILLUS   MALLEI. 

Historical. — Glanders  is  a  disease  primarily  of  animals  having  an 
undivided  hoof:  horses,  asses,  and  mules.  It  may  be  acute  or  chronic, 
and  two  clinical  types  are  recognized:  glanders,  an  initial  infection 
of  the  nasal  mucosa  and  regional  lymphatic  glands,  later  an  involve- 
ment of  the  internal  organs,  more  commonly  the  lungs;  and  farcy, 
a  cutaneous  glanders,  in  which  the  cutaneous  lymphatics  are  involved 
with  the  formation  of  nodules  (farcy  buds)  which  frequently  ulcerate 
and  discharge  a  cohesive  sticky  secretion.  Man  is  occasionally 
infected,  the  disease  being  one  of  the  most  fatal  known.  The  causa- 
tive organism,  Bacillus  mallei,  was  described  by  Loffler  and  Schiitz 
in  1882.1 

Morphology. — Bacillus  mallei  is  a  small  bacillus  with  rounded  or 
somewhat  attenuated  ends,  measuring  from  0.5  to  0.75  micron  in 
diameter,  and  from  2  to  5  microns  in  length.  The  organisms  occur 
singly  and  in  pairs  in  culture  media,  although  long  filamentous  forms 
are  not  uncommon  on  potato.  In  pus  and  from  tissues  the  bacilli 
occur  in  groups  or  clusters.  The  bacilli  frequently  appear  as  short, 
almost  coccoid  elements,  both  in  culture  and  in  vivo.  Older  cultures 
frequently  contain  many  branched  forms.  The  glanders  bacillus  is 
non-motile,  and  possesses  no  flagella.  Capsules  and  spores  have  not 
been  observed.  The  organism  stains  faintly  with  ordinary  anilin 
dyes^  better  with  those  having  an  alkaline  reaction.  It  is  Gram- 
negative  ^t  Stained  with  LofHer's  alkaline  methylene  blue,  the 
organism  exhibits  irregularity  of  colorable  material;  the  bacilli  may 
even  resemble  groups  of  cocci  with  faintly  stain  able  substance  con- 
necting the  deeply  stained,  round  granules.  Zeit2  has  called  atten- 
tion to  the  resemblance  of  B.  mallei  in  pus  and  tissue  to  staphylococci 
when  stained  with  methylene  blue,  and  the  possibility  of  error  in  diag- 

1  Deutsch.  med.  Wchnschr.,  1882,  No.  52. 

2  Jour.  Am.  Med.  Assn.,  1909,  lii,  181. 


368  GLANDERS— ANTHRAX— PYOCYANEUS 

nosis  upon  morphological  examination  alone.  The  Gram  stain  will 
distinguish  between  the  two,  however. 

Isolation  and  Culture. — Bacillus  mallei  grows  well  upon  ordinary 
laboratory  media,  better  if  glycerin  is  added,  and  upon  blood  serum 
and  potato.  The  first  growth  outside  the  animal  body  may  be  diffi- 
cult to  obtain.  Colonies  on  glycerin  agar  are  small,  yellowish  and 
round.  At  first  the  growths  are  translucent,  later  they  become  nearly 
opaque  and  more  deeply  colored.  Qrowth  in  gelatin  is  slow  and  not 
distinctive;  no  liquefaction  takes  place.  A  uniform  turbidity  appears 
in  broth  after  twenty-four  hours'  incubation  at  37°  C.,  which  gradually 
settles  out  as  a  tenacious,  slimy  sediment.  If  the  culture  is  undis- 
turbed, a  pellicle  gradually  forms  on  the  surface  of  the  medium. 
Litmus  milk  is  slowly  acidified,  and  coagulation  may  occur  after 
seven  to  fourteen  days'  incubation.  Growth  on  old  alkaline  potato 
is  distinctive;  after  twenty-four  to  forty-eight  hours'  incubation  a 
light  brown,  translucent  layer  appears,  which  has  been  likened  in 
color  and  general  appearance  to  a  layer  of  honey.  Later  the  growth 
becomes  darker,  even  brownish-red  in  color,  and  the  underlying  potato 
becomes  greenish  or  even  brown.  Potato  that  is  acid  does  not  exhibit 
the  typical  honey  yellow  growth. 

The  glanders  bacillus  is  aerobic,  facultatively  anaerobic;  the  opti- 
mum temperature  of  development  is  37°  C.,  growth  ceases  above  43° 
C.,  and  is  extremely  slow  below  25°  C.  The  resistance  of  the  organism 
to  chemical  agents  is  not  great,  but  it  remains  viable  for  several  weeks 
when  dried  in  pus  or  blood  and  maintained  in  a  cool,  dark  place.  An 
exposure  of  naked  bacilli  to  55°  C.  for  five  to  seven  minutes  kills  the 
organisms. 

Products  of  Growth. — Chemical. — Bacillus  mallei  is  culturally  inert 
in  purely  protein  media:  indol,  skatol  and  other  products  of  degrada- 
tion of  amino  acids  are  not  produced.  Acid,  but  no  gas,  is  formed  in 
dextrose  broth,  and  acid  is  produced  in  milk. 

Enzymes. — No  enzymes  have  been  demonstrated  in  cultures  of  B. 
mallei. 

Toxins. — Soluble  toxins  have  not  been  isolated  from  growths  of  the 
glanders  bacillus;  the  poison  of  the  organism  belongs  to  the  group 
of  the  endotoxins.  A  substance  analogous  to  tuberculin  has  been 
prepared  from  four  to  five  weeks'  glycerin  broth  cultures  of  Bacillus 
mallei,  mallein  or  morvin.  The  preparation  of  mallein  is  essentially 
the  same  as  for  tuberculin.  The  injection  of  mallein  in  moderate 
doses  into  normal  animals  may  lead  to  transient  fever  and  a  slight 


BACILLUS  MALLEI  369 

local  swelling  which  quickly  subsides.  In  horses  infected  with  B. 
mallei  a  swelling  appears  within  a  few  hours  which  is  painful  and 
inflamed ;  it  gradually  enlarges  for  twenty-four  hours  or  more,  and  the 
lymphatics  of  the  area  usually  become  prominent.  The  swelling  may 
persist  for  several  days,  but  gradually  diminishes  and  usually  disap- 
pears within  ten  days.  The  temperature  rises  with  the  local  swelling 
and  reaches  a  point  1°  to  2°  or  even  3°  above  the  normal  within  twenty- 
four  hours.  The  animal  usually  exhibits  all  the  signs  of  a  generalized 
reaction;  it  becomes  listless,  the  coat  roughens,  and  there  is  greater 
or  lesser  generalized  weakness.  The  temperature  usually  persists  for 
forty-eight  hours  or  more.  The  reaction  is  specific  but  requires 
experience  for  its  interpretation.  Variations  in  temperature  are 
caused  by  strangles,  .bronchitis  and  other  inflammatory  infections, 
hence  the  temperature  should  be  observed  for  some  hours  before  the 
injection  of  the  mallein.  A  positive  reaction  is  of  more  diagnostic 
value  than  a  negative  reaction.  It  should  be  borne  in  mind  that 
mallein  interferes  with  serologic  tests,  hence  the  latter  should  be  made 
before  the  injection  of  mallein*. 

Pathogenesis. — Animal. — Cattle  appear  to  be  immune  to  glanders; 
swine  are  but  slightly  susceptible;  cats,  sheep,  goats,  field  mice  and 
guinea-pigs'  are  susceptible,  but  white  mice  are  refractory. 

Acute  glanders  in  horses  and  asses  begins  after  an  incubation  period 
of  from  three  to  six  days  with  an  abrupt  rise  of  temperature  and  a 
viscid,  purulent  nasal  discharge.  The  nasal  mucosa,  at  first  deeply 
congested,  becomes  ulcerated;  the  regional  lymph  glands  enlarge 
and  may  suppurate.  The  lungs  become  involved  and  death  usually 
occurs  within  six  to  fourteen  days;  occasionally  the  animal  lives 
several  weeks.  The  onset  of  the  chronic  form  is  somewhat  more  insid- 
ious, and  the  symptoms  are  less  violent.  There  is  usually  a  nasal 
discharge  which  may  be  blood-streaked,  and  the  superficial  glands 
of  the  neck  are  palpable.  The  cutaneous  lymph  glands  and  usually 
the  lymph  channels  as  well  become  generally  enlarged,  and  they  may 
break  down  and  suppurate.  The  disease  may  run  a  very  mild  course, 
hardly  noticeable,  and  frequently  terminates  in  a  cure  after  months 
or  years. 

The  injection  of  material  from  ulcers,  nasal  secretion,  or  lymph 
glands  into  male  guinea-pigs  leads,  usually  within  two  or  three  days, 
to  a  characteristic  lesion,  unless  the  material  is  grossly  contaminated 
with  other  organisms,  namely,  a  purulent  orchitis;  the  testicle 
enlarges  until  it  can  not  be  retracted,  and  the  inflammation  spreads 

24 


370  GLANDERS— ANTHRAX— PYOCYANEUS 

from  the  tunica  vaginalis  to  the  epididymis.  The  peritoneum  is 
inflamed,  and  if  the  organism  is  not  very  virulent  there  is  joint 
involvement  and  gradual  emaciation  and  death.  This  is  known  as  the 
Straus  reaction. 

Human. — The  essential  lesion  in  man  is  similar  to  that  in  the  horse— 
a  granulomatous  nodule  made  up  chiefly  of  epithelioid  cells  and 
many  lymphoid  cells.  The  bacilli  occur  in  these  nodules  in  large 
numbers  as  a  rule.  The  nodules  occur  chiefly  in  the  nasal  mucosa, 
or  in  cutaneous  infections  under  the  skin;  they  break  down  readily, 
causing  ulceration  or  abscess  formation.  A  crop  of  papules,  which 
soon  break  down,  appears  on  the  face,  around  joints,  and  frequently 
upon  the  arms.  The  disease  terminates  fatally  in  about  65  to  70  per 
cent,  of  all  cases. 

Immunity  and  Immunization. — Recovery  from  an  attack  of  glanders 
does  not  appear  to  confer  immunity  to  subsequent  infection,  and 
attempts  to  induce  immunity  in  susceptible  animals  by  vaccines,  by 
the  use  of  mallein,  or  by  sera  have  been  unsuccessful.  Specific  agglu- 
tinins  and  precipitins  are  present  in  the  blood  serum  of  infected  animals 
and  a  diagnosis  can  be  made  by  the  method  of  complement  fixation. 
The  latter  procedure,  important  in  horses  and  other  domestic  animals, 
has  not  been  tried  very  extensively  in  man,  partly  because  of  the 
comparative  rarity  of  cases. 

Bacteriological  Diagnosis. — 1.  Microscopical  Examination. — Material 
from  the  purulent  discharges  of  the  nose  or  scrapings  from  cutaneous 
nodules  are  stained  by  Gram's  method  and  by  Loffler's  alkaline 
methylene  blue.  The  organism  is  Gram-negative,  and  frequently 
exhibits  a  beaded  appearance  not  unlike  the  diphtheria  bacillus.  A 
diagnosis  based  upon  purely  morphological  characters  is  not  reliable. 

2.  Cultural. — Scrapings  from  unopened  granulomata  or  from  the 
organs   postmortem   should   be   inoculated   upon   potato   having   an 
alkaline  reaction.    The  characteristic  appearance  of  the  growth  upon 
this  medium  is  suggestive",  but  not  conclusive.     Bacillus  pyocyaneus 
grows  very  similarly.    Pus  must  be  plated  upon  glycerin  agar  or  blood 
serum,  because  the  discharges  from  ulcers  and  abscesses  are  almost 
invariably  contaminated  with  other  organisms.     Pure  cultures  are 
examined  microscopically  and  injected  into  male  guinea-pigs  intra- 
peritoneally. 

3.  Animal  Injection. — The  intraperitoneal   injection  of  suspected 
material  into  the  peritoneal  cavity  of  male  guinea-pigs  leads,  in  the 
absence  of  organisms  capable  of  causing  a  violent  peritonitis,  to  the 


BACILLUS  MALLEI  371 

localization  of  the  bacilli  in  the  testes,  which  become  inflamed  and 
swollen — the  Straus  reaction.  The  animal  usually  dies  within  a 
week.  Potato  cultures  and  microscopical  examination  of  the  purulent 
material  in  the  testes  usually  .suffices  to  establish  the  diagnosis.  In 
case  the  material  for  examination  is  contaminated  with  other  bacteria, 
it  is  advisable  to  inoculate  it  into  the  subcutaneous  tissues  of  one 
guinea-pig,  and  to  inoculate  a  second  male  pig  with  material  from 
an  enlarged  lymph  gland  of  the  first  pig.  A  negative  examination 
is  inconclusive. 

Serological  Diagnosis. — (a)  Mallein. — Discussed  above. 

(6)  Ophthalmo  Reaction. — The  instillation  of  a  few  drops  of  mallein 
into  the  conjunctival  sac  of  a  glanderous  horse  leads  to  a  reaction 
very  similar  to  the  ophthalmo-tuberculin  reaction  in  man,  except 
that  in  positive  cases  a  purulent  discharge  as  well  as  a  red  inflamed 
conjunctiva  results. 

(c)  Agglutination  Test. — Specific  agglutinins  for  Bacillus  mallei 
appear  in  the  blood  of  infected  animals  usually  within  four  to  seven 
days  in  acute  glanders,  and  there  is  a  rough  parallelism  between  the 
severity  of  the  disease  and  the  development  of  the  immune  bodies. 
The  agglutinins  as  a  rule  diminish  considerably  if  the  disease  becomes 
chronic,  and  may  become  reduced  to  such  a  degree  that  the  reaction 
becomes  unreliable.  The  sera  of  normal  horses  frequently  contain 
non-specific  agglutinins  which  may  clump  glanders  bacilli  in  dilutions 
of  1  to  100  to  1  to  300.  Injections  of  mallein  appear  to  influence 
antibodies  specific  for  the  glanders  bacillus  adversely,  consequently 
serological  examinations  should  be  made  before  mallein  is  injected. 

Serum  for  agglutination  tests  should  be  withdrawn  in  a  sterile  syringe 
from  the  jugular  vein  in  the  horse,  and  from  the  median  basilic  vein 
in  man.  The  serum,  separated  from  the  clot,  is  diluted  with  a  suspen- 
sion of  glanders  bacilli  to  the  following  degrees:  1  to  500,  1  to  1000, 
1  to  2500,  1  to  5000,  1  to  7500.  Glanders  bacilli,  virulent  for  guinea- 
pigs  (obtained  by  passing  glanders  bacilli  through  a  series  of  animals 
until  the  organism  kills  the  animal  within  five  days — intraperitoneal 
injection),  from  glycerin  agar  slants  are  emulsified  in  physiological 
salt  solution  containing  0.5  per  cent,  carbolic  acid,  thoroughly  shaken 
and  filtered  through  a  thin  layer  of  absorbent  cotton  to  remove  clumps. 
Salt-phenol  solution  is  added  to  the  suspension  until  a  moderately 
turbid  suspension  is  obtained.  Decreasing  amounts  of  serum  from 
the  suspected  animal  are  added  to  obtain  the  dilutions  mentioned 
above.  A  normal  serum  and  a  known  positive  serum  are  diluted  in 


372  GLANDERS— ANTHRAX— PYOCYANEUS 

the  same  manner  to  serve  as  controls.  Incubation  is  continued  at 
37°  C.  for  seventy-two  hours,  because  the  reaction  is  usually  slow  in 
developing.  Sterility  must  be  maintained  throughout.  Strongly 
positive  sera  may  give  a  definite  clumping  in  twenty-four  hours  or 
less;  the  supernatant  fluid  becomes  clear,  and  the  organisms  collect 
as  a  diffuse  sediment  at  the  bottom  of  the  tube.  A  negative  reaction 
is  indicated  by  a  turbid  supernatant  fluid.  The  reaction  may  be 
made  microscopically  or  macroscopically,  the  latter  being  preferable. 

Attempts  have  been  made  to  shorten  the  reaction  time  by  aiding 
sedimentation  with  the  centrifuge.  The  various  dilutions  are  incu- 
bated for  a  full  hour  at  37°  C.,  allowing  fifteen  minutes  for  the  tubes 
to  reach  37°  C.  in  the  incubator;  then  they  are  whirled  for  fifteen 
minutes  at  a  speed  with  a  twenty-four  inch  radius  not  exceeding  1500 
revolutions,  placed  in  the  ice-box  and  examined  after  three  hours. 
The  slowly  developing  reactions  may  not  be  definitely  positive  for 
twenty-four  hours. 

A  reaction  in  a  dilution  of  1  to  500  (horse,  ass  or  mule)  is  the  lowest 
limit  to  which  a  definite  reaction  may  be  attributed,  and  the  result 
should  be  controlled  with  a  mallein  test.  Dilutions  of  1  to  750  or 
higher  are  usually  safely  regarded  as  diagnostic.  In  human  cases  a 
positive  reaction  in  a  dilution  of  1  to  100  is  diagnostic. 

The  method  of  complement  fixation  (see  page  164  for  details)  is 
rapidly  becoming  a  general  method  for  the  diagnosis  of  glanders. 

Dissemination  and  Prophylaxis. — Glanders  is  transmitted  by  direct 
contact,  by  infection  through  cutaneous  abrasions  and  cuts,  and  by 
feeding  paraphernalia,  watering  troughs  and  buckets.  In  man  cuta- 
neous infection  is  more  common. 

BACILLUS   ANTHRACIS. 

Bacillus  anthracis  was  first  seen  by  Davaine1  in  1863,  in  the  blood 
of  animals  infected  with  anthrax.  Koch2  confirmed  Davaine 's  obser- 
vation, obtained  the  organism  in  pure  culture,  and  reproduced  the 
disease  with  these  cultures  in  other  animals,  thus  establishing  the 
etiology  of  anthrax.  He  also  demonstrated  spore  formation  by  B. 
anthracis  upon  artificial  media. 

Morphology. — Bacillus  anthracis  is  a  rod-shaped  organism  measur- 
ing from  1  to  1.50  microns  in  diameter  and  from  2  to  4  microns  in 

1  Compt.  rend.  Acad.  Sci.,  1863,  Ivii. 

2  Cohn's  Beitr.  z.  Biol.  der  Pflanzen,  1876,  ii,  277. 


BACILLUS  ANTHRACIS  373 

length.  Occasionally  filaments  20  to  25  microns  in  length  are 
observed,  which  exhibit  no  demonstrable  septation;  these  long  rods 
may  be  single  cells  or  chains  of  cells  in  which  septation  is  imper- 
fect. The  ends  of  the  bacilli  are  square  cut  and  often"  appear 
to  be  concave,  particularly  when  the  organisms  are  examined  in  a 
strained  preparation  made  directly  from  the  blood  of  an  infected 
animal.  Occasionally  the  ends  are  somewhat  thickened,  giving  the 
bacillus  an  appearance  which  suggests  a  segment  of  bamboo.  Bacillus 
anthracis  produces  short  chains  of  three  to  eight  elements  in  the 
bloodvessels  of  infected  animals,  and  in  artificial  media  it  produces 
long,  coiled  chains  of  bacilli  which  give  a  characteristic  filamentous 
appearance  to  the  colonies  upon  solid  media.  The  organism  is  non- 
motile,  and  possesses  no  flagella.  A  capsule1  is  formed  around  the 


FIG.  52. — Bacillus  anthracis,  spore  formation.      X  1000.     (Gunther.) 


bacilli  in  the  animal  body  and  also  in  cultures  containing  albuminous 
substances,  as  uncoagulated  blood  serum.2  Spores  are  produced  in 
media  freely  exposed  to  the  air  between  the  temperatures  of  15°  C.  and 
40°  C.  The  lower  limit  of  spore  formation  has  a  practical  bearirtg 
upon  the  presence  of  anthrax  spores  in  soil.  In  the  temperate  zones 
a  temperature  exceeding  15°  C.  in  midsummer  is  not  found  at  depths 
greater  than  five  feet,  hence  anthrax  carcasses  buried  deeply  are  not 
likely  to  cause  infection  of  the  soil.  It  has  been  stated  that  earthworms 
may  carry  infected  material  from  the  deeper  layers  of  the  soil  to  the 

1  The  capsule  was  first  seen  by  Serafini  (Progress©  Medico,  1888),  but  Johne  (Deutsch. 
Ztschr.  f.  Tiermed.  u.  vergl.  Path.,  1893,  xix,  244;  1894,  xx,  426)  first  called  attention 
to  the  diagnostic  importance  of  the  capsule  in  the  diagnosis  of  anthrax  of  the  domestic 
animals. 

2Haase,  Deutsch.  Ztschr.  f.  Tiermed.  u.  vergl.  Path.,  1894,  xx,  429;  Johne,  ibid., 
1894,  xxi,  142. 


374  GLANDERS— ANTHRAX— PYOCYANEUS 

surface,  where  speculation  may  occur.  If  these  temperatures  are 
exceeded  in  either  direction,  spore  formation  does  not  occur.  The 
spores,  which  are  oval,  are  situated  at  or  near  the  centre  of  the  cell 
and  measure  about  0.8  micron  in  diameter  and  from  1.2  to  1.4  microns 
in  length.  Occasional  asporous  strains1  are  met  with,  and  spore 
formation  may  be  suppressed  by  cultivating  the  bacteria  at  42°  C. 
for  several  hours  or  in  fluid  media  containing  potassium  bichromate 
in  dilutions  from  1  to  5000  to  1  to  2000,  or  small  amoimts  of  phenol.2 
Lehmann3  states  that  long-continued  transfer  of  cultures  from  gelatin 
to  gelatin  frequently  leads  to  a  suppression  of  spore  formation.  Some 
strains  become  asporeless  much  more  readily  than  others.4  Spores 


FIG.  53. — Bacillus  anthracis,  showing  capsule  formation.     X  1000.    (Kolle  and  Hetsch.) 


are  not  formed  in  the  intact  animal  body.  Mature  vegetative  bacilli 
emerge  from  the  spores  in.  the  presence  of  oxygen,  if  the  temperature 
is  maintained  between  15°  and  40°  C.  The  spore  membrane  merges 
imperceptibly  into  the  newly  formed  vegetative  cell;  no  visible  rup- 
turing of  the  spore  membrane  is  detectable. 

Bacillus  anthracis  stains  well  with  ordinary  anilin  dyes  and  young 
cultures  are  Gram-positive.  Older  cultures  may  gradually  lose  their 
ability  to  retain  Gram's  stain.  Spores  may  be  stained  with  the  Ziehl- 
Neelsen  stain.  (See  Staining  of  Spores.) 

Isolation  and  Culture. — Bacillus  anthracis  grows  readily  upon  any 
artificial  media.  Material  is  best  obtained  from  the  spleen  or  liver 

1  Asporous  cultures  do  not  necessarily  become  avirulent  (Chamberland  and  Roux, 
Compt.  rend.  Acad.  des  Sci.,  1883,  xcvi,  1090). 

2  Roux,  Ann.  Inst.  Past.,  1890,  25. 

3  Milnchen.  med.  Wchnschr.,  1887,  No.  26. 

4  Surmont  and  Arnould,  Ann.  Inst.  Past.,  1894,  p.  832. 


BACILLUS  ANTHRACIS        .  375 

of  dead  animals,  or  from  the  blood  of  an  infected  animal.  Gelatin 
is  rapidly  liquefied;  colonies  appear  in  gelatin  plates  within  eighteen 
hours  after  inoculation,  which  are  from  1  to  2  mm.  in  diameter.  They 
are  gray,  opaque,  and  somewhat  irregular  in  size.  The  organisms 
develop  rapidly,  and  liquefaction  commences  within  thirty  hours  as  a 
rule.  At  this  stage  of  development  the  edges  of  the  colonies  are  com- 
posed of  tangled,  radiating  chains  of  bacilli  which  extend  into  the 
surrounding  medium,  and  the  colony  itself  is  composed  of  a  mass  of 
twisted  filaments  which  has  been  likened  to  a  Medusa  head.  Few, 
if  any,  pathogenic  bacteria  present  such  an  appearance.  The  growth 
in  stab  cultures  in  gelatin  is  also  characteristic;  the  organisms  grow 


FIG.  54. — Bacillus  anthracis,  section  from  kidney,  semi-diagrammatic.      X  500. 
(Kolle  and  Hetsch.) 

away  from  the  line  of  inoculation  into  the  medium  as  spikelets  which 
resemble  an  "inverted  pine  tree."  Liquefaction  soon  takes  place. 
Milk  is  rendered  acid,  and  the  casein  precipitated  and  slowly  liquefied. 
A  pellicle  forms~~upon  the  surface  of  broth  which  readily  becomes 
detached  from  the  sides  of  the  tube  and  settles  to  the  bottom.  No 
turbidity  is  produced  in  fluid  media. 

Bacillus  anthracis  is  a  strongly  aerobic  bacillus,  but  growth  will 
take  place  under  anaerobic  conditions.  Growth  is  very  slow  at  18° 
C.,  and  ceases  below  15°  C.  The  optimum  is  about  37°  C.,  and 
development  does  not  take  place  at  45°  C. 

The  vegetative  (asporeless)  organisms  are  not  resistant  to  heat  or 
drying.  The  spores  are  very  resistant.  Dried  spores  have  remained 
viable  and  virulent  for  eighteen  years.1  Fresh  blood  containing  anthrax 

1  V.  Szekely,  Ztschr.  f.  Hyg.,  1903,  xliv,  363. 


376  GLANDERS— ANTHRAX— PYOCYANEUS 

bacilli  may  remain  viable  for  two  months  if  relatively  thick  layers 
are  prepared.  Dry  heat  at  160°  C.  kills  anthrax  spores  within  one 
and  a  half  hours;  live  steam  (100°  C.)  kills  them  within  ten  minutes. 
Carbolic  acid  is  not  very  effective  as  a  germicide,  but  1  to  1000  bichlo- 
ride of  mercury  kills  the  spores  within  half  an  hour.  Direct  sunlight 
kills  them  within  six  hours.1 

Products  of  Growth. — Chemical. — Martin2  found  protoalbumose , 
deuteroalbumose,  a  trace  of  peptone,  an  alkaloidal  substance,  and 
small  amounts  of  leucin  and  tyrosin  in  a  serum  culture  of  B.  anthracis. 
Nojacid  or  gas  is  produced  in  any  sugar  media.  The  albumoses  and 
peptone  caused  a  febrile  reaction  in  animals,  and  the  alkaloidal  sub- 
stance (anthrax-alkaloid)  caused  edema  and  congestion.  These  results 
have  never  been  repeated.3 

Enzymes. — Bacillus  anthracis  produces  a  proteolytic  enzyme  which 
liquefies  gelatin,  blood  serum  and  casein.  No  other  enzymes  are 
known. 

Toxins. — Soluble  toxins  have  not  been  demonstrated  in  cultures 
of  anthrax  bacilli,  and  the  nature  of  the  endotoxin  is  unknown — the 
cellular  substance  of  the  organism  is  not  as  toxic  as  that  of  many  other 
pathogenic  bacteria,  and  the  nature  of  the  action  of  the  bacillus  is 
not  clearly  determined. 

Pathogenesis. — Animal. — Anthrax  is  a  disease  of  cattle,  sheep4  and 
horses.  Swine  are  less  susceptible.  Guinea-pigs,  rabbits  and  white 
mice  are  very  susceptible  to  inoculation.  Rats  and  dogs  succumb  to 
large  doses.  Birds  and  cold-blooded  animals  are  naturally  immune, 
although,  as  Pasteur  showed,  the  immunity  may  be  overcome  by 
reducing  the  body  temperature  of  birds  and  by  raising  the  body  tem- 
perature of  cold-blooded  animals. 

The  artificially-induced  disease  in  small  laboratory  animals  is 
usually  a  rapidly  fatal  septicemia;  the  organisms  swarm  in  the  blood- 
vessels and  appear  upon  section  to  almost  occlude  the  capillaries. 
The  spleen  is  greatly  enlarged  and  there  is  congestion  of  the  other 
glandular  organs.  Cattle  and  sheep  readily  succumb  to  infection 
with  pure  cultures  of  the  organism.  The  natural  infection  in  cattle 
and  sheep  appears  to  be  chiefly  through  the  intestinal  tract.  In  horses 

1  Moment,  Ann.  Inst.  Past.,  1892,  23. 

2  Proc.  Royal  Soc.,  London,  May  22,  1890.     Brit.  Med.  Jour.,  March  26,  April  2,  9, 
1892.     Animal  Report  Local  Government  Board,  Supplement,  1890-91,  xx,  255-266. 

3  It  is  probable  that  these  substances  were  produced  from  the  serum  by  the  action  of 
the  organism ;  they  cannot  be  regarded  as  specific  toxic  products. 

4  Algerian  sheep  are  said  to  be  more  resistant  to  infection  than  ordinary  sheep. 


BACILLUS  ANTHRACIS  377 

infection  may  take  place  through  the  skin  as  well.  Less  commonly 
cutaneous  infection  may  occur  through  wounds  in  cattle  and  sheep. 
A  localized  severe  inflammation  results  which  may  heal  spontaneously 
or  lead  to  a  generalized  infection.  It  is  stated  that  flies,  particularly 
the  horse  flies  (Tabanidse)  may  transmit  the  virus  to  animals.  The 
disease  may  also  be  transmitted  experimentally  by  the  inhalation  of 
spores;  this  method  of  infection  is  probably  not  common  in  animals. 

Human. — Anthrax  bacilli  or  their  spores  may  cause  disease  in  man 
either  by  gaining  entrance  to  the  body  through  abrasions  of  the  skin, 
by  inhalation,  or  by  ingestion.  Inoculation  through  the  skin  may 
give  rise  to  malignant  pustule,  characterized  by  a  small  papule  at  the 
site  of  infection,  which  soon  becomes  vesicular.  The  process  may 
stop  spontaneously  with  the  formation  of  a  scab  and  the  gradual 
drying  up  of  the  vesicle,  or  the  inflammation  may  spread,  producing 
a  wide  area  of  induration  in  which  vesicles  appear,  often  in  consider- 
able numbers.  The  involved  area  becomes  edematous,  and  the 
regional  glands  become  enlarged.  Death  may  ensue  within  five  to 
seven  days,  or  the  inflamed  area  slowly  returns  to  normal.  Less  com- 
monly edema  is  the  prominent  symptom,  pustule  formation  being 
absent  or  not  conspicuous.  The  edematous  area  spreads  rapidly  and 
it  may  be  extensive  enough  to  interfere  with  the  nutrition  of  the  part 
and  lead  to  gangrene.  The  head,  the  arms,  or  the  hands  are  more 
frequently  involved  than  the  lower  extremities. 

Intestinal  anthrax  and  pneumonic  anthrax  or  woolsorters'  disease 
are  usually  caused  by  the  ingestion  or  inhalation  of  anthrax  bacilli 
or  their  spores.  Intestinal  anthrax  is  uncommon;  it  is  supposed  to 
be  an  infection  through  the  gastro-intestinal  tract  resulting  from  the 
ingestion  of  meat  or  milk  of  diseased  animals.  The  symptoms  are 
essentially  those  of  meat-poisoning:  chill,  vomiting,  and  nausea, 
diarrhea,  and  some  .fever.  Woolsorters'  disease  prevails  where  hides 
and  wool,  particularly  from  South  America,  Morocco  and  Russia,  are 
handled.  The  symptoms  are:  a  sudden  chill,  immediate  great  pros- 
tration, intense  pain,  bronchial  irritation,  and  occasionally  death 
within  twenty-four  hours.  Cerebral  symptoms  frequently  are  promi- 
nent in  those  cases  which  are  more  protracted.  There  are  no  distinc- 
tive postmortem  changes;  the  lungs  may  be  edematous  and  there 
are  scattered  patches  of  lobular  pneumonia  with  inflammation  of  the 
regional  bronchi. 

Immunity  and  Immunization. — The  vulnerability  of  human  tissues 
to  anthrax  infection  is  varied;  the  skin  appears  to  be  relatively  resis- 


378  GLANDERS— ANTHRAX— PYOCYANEUS 

tant,  but  the  lungs  are  very  susceptible.  The  disease  resulting  from 
infection  of  the  lungs  by  anthrax  bacilli  is  one  of  the  most  rapid  and 
fatal  known  to  man.  Practically  no  attempt  has  been  made  to 
immunize  man  to  anthrax,  but  Sobernheim  has  prepared  a  serum 
obtained  by  injecting  animals  immunized  by  Pasteur's  method  with 
virulent  anthrax  bacilli,  which  is  said  to  be  of  some  value  as  a  curative 
agent  in  malignant  pustule. 

Animal  Immunization. — Pasteur  protected  animals  against  anthrax 
infection  by  vaccination  with  attenuated  anthrax  bacilli.  Two  vac- 
cines were  used;  they  were  prepared  in  the  following  manner:  Vaccine 
A  was  obtained  by  growing  anthrax  bacilli  at  42.5°  C.  for  six  weeks. 
The  organisms  are  asporeless  after  this  treatment,  but  they  grow 
luxuriantly.  They  are  avirulent  for  rabbits  and  guinea-pigs,  but  kill 
mice.  Vaccine  B  was  obtained  by  growing  anthrax  bacilli  at  42.5° 
C.  for  two  weeks.  The  organisms  kill  mice  and  guinea-pigs,  but  do 
not  kill  rabbits.  Vaccine  A  is  injected,  and  after  two  weeks  Vaccine 
B  is  injected,  both  subcutaneously.  The  animals  are  immune  two 
weeks  after  the  last  injection  to  cutaneous  infection  with  anthrax 
bacilli,  but  are  somewhat  less  resistant  to  infection  by  way  of  the 
alimentary  tract.  The  immunity  is  of  about  one  year's  duration, 
and  it  must  be  renewed  at  the  end  of  that  time.  Sobernheim1  has 
attempted  to  increase  the  immunity  to  ingestion  anthrax  by  injecting 
his  serum  (5  to  15  c.c.)  and  Vaccine  B  of  Pasteur  simultaneously.  He 
states  that  this  combined  immunizing  process  brings  the  resistance 
of  the  animal  to  such  a  level  that  ingestion  infection  rarely  or  never 
occurs. 

Bacteriological  Diagnosis. — The  diagnosis  of  anthrax  in  man  depends 
wholly  upon  the  identification  of  the  anthrax  bacillus. 

(a)  Morphological  Diagnosis. — Smears  from  the  blood  or  tissues  of 
animals  stained  by  Gram's  method  show  large,  square-ended,  Gram- 
positive  bacilli,  which  occur  singly,  in  pairs,  or  short  chains.     The 
organisms  are  encapsulated  but  require  special  capsule  stains  for  their 
demonstration.     In  man  similar  examination  is  made  from  the  serous 
fluid  expressed  from  the  malignant  pustule,  the  blood  (best  obtained 
from  the  ear),  fluid  from  edematous  areas,  sputum  from  woolsorters' 
disease,  and  feces  from  intestinal  cases. 

(b)  Cultural. — The  material  collected  aseptically  is  inoculated  into 
ordinary  media.     It  is  well  to  examine  the  media  after  two  to  three 
days'  incubation  for  spores  if  the  culture  is  impure;    if  spores  are 

1  Ztschr.  f.  Hyg.,  1899,  xxxi,  89. 


BACILLUS  PYOCYANEVS  379 

found  heating  the  culture  to  80°  C.  for  fifteen  minutes  will  destroy  all 
vegetative  forms  leaving  the  anthrax  spores  in  excess  and  frequently 
in  pure  culture.  The  growth  on  gelatin  is  fairly  distinctive. 

(c)  Inoculate  a  guinea-pig  or  a  mouse  with  a  small  amount  of  blood 
or  fluid  from  a  suspected  lesion;  if  bacilli  are  not  numerous,  incubate 
the  material  in  broth  for  twenty-four  hours,  then  inject  the  enriched 
culture.  The  occurrence  of  typical  large  Gram-positive  bacilli  in  the 
blood  stream  postmortem  is  sufficient  to  establish  the  diagnosis  in 
the  light  of  the  clinical  history.  The  principal  organisms  likely  to 
cause  confusion  are:  B.  subtilis  and  members  of  the  mesentericus 
group,  which  do  not  produce  acute  death  in  guinea-pigs  by  generalized 
septicemia,  and  B.  edematis  maligni  and  B.  aerogenes  capsulatus, 
both  of  which  are  obligate  anaerobes. 

Dissemination. — The  spores  of  anthrax  bacilli  are  extremely  resis- 
tant to  dessication,  and  they  remain  alive  for  years  in  the  soil.  Once 
a  pasture  or  other  enclosure  is  infected  with  the  organisms  it  is  unsafe 
to  permit  cattle,  sheep  or  other  domestic  animals  to  graze  there. 
The  washings  from  such  infected  lands  may  convey  infection  to  other 
lands. 

Prophylaxis  in  man  consists  essentially  in  preventing  contact  infec- 
tion with  diseased  animals  or  infected  material,  and  particular  care 
in  preventing  the  inhalation  of  dust  from  hides  or  wool  of  cattle  or 
sheep  from  countries  where  the  disease  is  prevalent;  this  applies  par- 
ticularly to  South  American,  Moroccan  and  Russian  hides  and  wool. 

BACILLUS   PYOCYANEUS. 

Historical. — Surgeons  for  many  years  have  noticed  that  occasional 
suppurating  wounds  discharge  pus  which  stains  bandages  a  green  or 
green-blue  color.  Gessard1  demonstrated  the  specific  organism,  Bacil- 
lus pyocyaneus,  in  pure  culture  and  described  it  in  considerable  detail. 
Somewhat  later  Charrin2  studied  the  pathogenesis  of  the  organism  for 
rabbits  (maladie  pyocyanique),  setting  forth  clearly  the  importance 
of  the  bacillus  as  a  disease-producing  microorganism. 

Morphology. — Bacillus  pyocyaneus  is  a  moderate-sized  organism 
with  rounded  ends,  usually  occurring  singly  or  in  pairs,  less  commonly 
in  short  chains.  The  dimensions  vary  considerably  even  in  the  same 
culture;  the  diameter  averages  about  0.6  micron,  although  some 

1  These  de  Paris,  1882. 

2  La  maladie  pyocyanique,  Paris,  1889. 


380  GLANDERS— ANTHRAX— PYOCYANEUS 

bacilli  measure  but  0.3  micron  and  others  as  much  as  1  micron.  The 
length  varies  between  1.5  and  4  microns,  the  average  being  about  2 
microns.  The  organism  is  actively  motile,  and  possesses  a  terminal 
polar  flagellum  (monotrichic  flagellation).  Capsules  and  spores  have 
not  been  observed.  Ordinary  anilin  dyes  color  the  bacillus  with  mod- 
erate intensity,  and  it^  is  Gram-negative,  although  the  gentian  violet 
is  somewhat  less  readily  removed  by  alcohol  than  from  a  majority 
of  Gram-negative  bacilli,  as  Bacillus  coli  for  example. 

Isolation  and  Culture. — The  organism  grows  readily  and  rapidly 
upon  ordinary  artificial  media,  producing  the  characteristic  pigments 
in  the  presence  of  oxygen.  The  colonies  on  agar  are  round  and  measure 
from  1  to  3  mm.  in  diameter  after  eighteen  to  twenty-four  hours' 
incubation  at  37°  C.  The  growth  spreads  rapidly,  and  the  pigment 
which  becomes  visible  within  eighteen  hours  dissolves  in  the  medium 
imparting  a  blue-green  color  to  it.  Gelatin  colonies  are  not  charac- 
teristic in  outline,  but  rapidly  liquefy  the  medium,  which  becomes 
green.  A  turbidity  is  visible  within  eight  hours  in  broth  and  a  pellicle 
usually  forms  on  the  surface.  A  viscous,  gray-brown  sediment  collects 
at  the  bottom  of  the  tube,  and  an  ammoniacal  odor  is  noticeable  even 
within  twenty-four  hours.  The  medium,  particularly  the  upper  layers 
in  contact  with  oxygen,  becomes  blue-green.  Milk  is  coagulated, 
the  coagulum  being  slimy,  and  eventually  partly  or  even  completely 
dissolved;  the  medium,  at  first  yellowish,  becomes  green,  then  blue, 
particularly  in  the  Upper  layers. 

Bacillus  pyocyaneus  is  aerobic,  facultatively  anaerobic.  The  opti- 
mum" temperature  is  37°  to  38°  C.;  development  is  sluggish  below  18° 
C.  and  practically  ceases  at  43°  to  44°  C. 

Products  of  Growth. — Chemical. — The  organism  produces  a  relatively 
large  amount  of  ammonia  from  pr.oteins  and  protein  derivatives,1  and 
in  milk.2 

Pigments. — Two  pigments  are  produced  by  Bacillus  pyocyaneus: 
a  water-soluble,  green,  fluorescent  pigment  similar  in  physical  proper- 
ties to  that  found  in  cultures  of  other  fluorescent  bacteria;  and  a 
specific  pigment,  pyocyanin,  which  is  insoluble  in  water  but  soluble 
in  chloroform.  Pyocyanin,  to  which  the  empirical  formula  Ci4Hi4NO2 
has  been  ascribed  by  Ledderhose,3  crystallizes  from  chloroform  solu- 


1Armaud  and  Charrin,  Compt.  rend.  Ac.  sc.,  1891,  cxii,  755,  1157;    Kendall,  Day, 
and  Walker,  Jour.  Am.  Chem.  Soc.,  1913,  xxxv,  1243. 

2  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Soc.,  1914,  xxxvi,  1948,  1963. 

3  Deutsch.  Ztschr.  f.  Chir.,  1888,  xxviii,  201. 


BACILLUS  PYOCYANEUS  381 

tion  as  blue  needles.  It  forms  salts  with  acids,  and  exists  as  a  leuco- 
base  in  cultures  from  which  oxygen  is  excluded.  The  color  changes 
to  a  brownish-red  in  old  cultures. 

Enzymes. — One  of  the  noteworthy  products  of  Bacillus  pyocyaneus 
is  a  soluble  proteolytic  enzyme,  a  protease,  which  dissolves  gelatin, 
casein,  coagulated  blood  serum  and  fibrin.1  Breymann2  showed  that 
the  bodies  of  the  bacteria,  freed  from  culture  media,  contained  the 
same  or  a  similar  enzyme.  Emmerich  and  Low3  isolated  a  proteolytic 
enzyme,  called  by  them  pyocyanase,  which  possessed  the  remarkable 
property  of  dissolving  alien  bacteria.  This  enzyme  has  been  used 
therapeutically  with  some  success.  Whether  pyocyanase  is  identical 
with  the  protease  mentioned  above  has  never  been  clearly  determined. 

No  diastatic  enzymes  have  been  detected  in  cultures  of  Bacillus 
pyocyaneus.4 

Toxins. — Wassermann5  found  that  filtered  cultures  of  Bacillus 
pyocyaneus  or  cultures  killed  with  toluol  would  kill  guinea-pigs  when 
injected  intraperitoneally  in  amounts  of  0.2  to  0.5  c.c.  The  organisms 
themselves  were  decidedly  less  toxic.  The  toxicity  is  not  attributable 
to  the  specific  pigment,  pyocyanin,  but  to  substances  of  unknown 
composition. 

Pathogenesis. — Animal. — Bacillus  pyocyaneus  is  pathogenic  for 
small  laboratory  animals,  guinea-pigs  being  the  most  susceptible. 
A  cubic  centimeter  or  less  of  an  actively  growing  broth  culture  intro- 
duced into  the  peritoneal  cavity  causes  death  within  twenty-four  hours 
as  a  rule.  There  is  edema,  leukocytosis,  and  the  peritoneal  fluid 
increased  in  amount  swarms  with  the  bacilli.  Rabbits  are  less  sus- 
ceptible; rats  and  mice  are  relatively  refractory.  The  subcutaneous 
injection  of  cultures  of  the  organism,  especially  if  the  virulence  is  not 
great,  leads  to  a  chronic,  wasting  infection  which  usually  terminates 
fatally.  The  subcutaneous  tissue  becomes  edematous  and  necrotic, 
and  ulceration  frequently  occurs. 

Human. — Besides  the  focal  lesions,  abscesses,  ulcers,  otitis  media, 
less  commonly  liver  abscesses,  and  bronchopneumonia,  Bacillus  pyo- 
cyaneus occasionally  produces  severe  gastro-intestinal  infection, 
especially  in  young  children,  generalized  sepsis,  and  inflammation  of 
serous  surfaces,  the  pleura,  pericardium,  and  peritoneum. 

1  Jakowski,  Ztschr.  f.  Hyg.,  1893,  xv,  474;  Fermi,  Centralbl.  f.  Bakt.,  1891,  x,  401; 
Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Soc.,  1914,  xxxvi,  1966,  and  others. 

2  Centralbl.  f.  Bakt.,  Orig.,  1902,  xxxi,  481. 

3  Ztschr.  f.  Hyg.,  1899,  xxxi,  1. 

4  Fermi,  loc.  cit.  s  Ztschr.  f.  Hyg.,  1896,  xxii,  263. 


382  GLANDERS— ANTHRAX— PYOCYANEUS 

Immunity  and  Immunization. — It  is  possible  to  immunize  animals 
both  by  the  cautious  injection  of  the  bacilli  which  stimulate  the  for- 
mation of  specific  bacteriolysins,  and  by  filtrates  of  broth  cultures  of 
the  organisms,  which  incite  the  formation  not  only  of  bacteriolytic 
substances,  but  antitoxic  substances  as  well.  No  practical  use  is 
made  of  these  antibodies  in  human  infections,  however. 

Bacteriological  Diagnosis. — Wounds  infected  by  Bacillus  pyocyaneus 
are  usually  diagnosed  by  the  blue-green  color  of  the  dressings.  The 
bacilli  are  readily  isolated  upon  gelatin  plates,  where  the  development 
of  the  blue-green  color  is  very  characteristic. 

BACILLUS   ABORTUS. 

Historical. — Infectious  abortion  is  a  disease  which  has  for  many 
years  been  recognized  as  an  important  economic  one  in  the  cattle 
industry.  Later  it  was  found  that  the  same  disease  also  exists  among 
horses,  goats  and  sheep.  The  organism  was  first  isolated  by  Bang.1 

Morphology. — B.  abortus  is  a  small  pleiomorphic  bacillus,  measuring 
0.4  to  0.6  micron  in  diameter,  by  0.6  to  2.5  microns  in  length.  It 
occurs  singly  and  in  pairs;  rarely  short  chains  of  three  to  six  elements 
are  found.  The  shape  varies:  some  organisms  are  almost  spherical, 
others  are  distinctly  rod-shaped,  the  latter  being  more  frequently 
found  in  broth  cultures  and  in  vivo.  According  to  Priesz,2  branched 
forms  may  be  found  in  older  cultures.  It  is  non-motile  and  possesses 
no  flagella,  although  Brownian  movement  may  be  fairly  active.  It 
possesses  no  capsules,  and  no  spores  have  been  demonstrated.  It 
stains  readily  with  ordinary  anilin  dyes,  but  somewhat  irregularly, 
some  areas  staining  more  intensely  than  others.  Occasionally  with 
the  methylene  blue  stain  the  organisms  may  present  a  bipolar  appear- 
ance. The  organism  is  Gram-negative. 

Isolation  and  Culture. — Initial  growths  on  artificial  media  outside 
the  animal  body  are  somewhat  difficult  to  obtain.  The  organism 
appears  to  grow  best  in  a  somewhat  rarified  atmosphere.  This  has 
been  obtained  by  Fabyan3  by  growing  the  organism  on  an  agar  slant 
which  is  connected  by  a  narrow  tube  with  an  agar  slant  on  which 
B.  subtilis  is  growing.  B.  subtilis  appears  to  so  change  the  percentage 
composition  of  the  air  in  the  two  tubes  that  B.  abortus  grows  fairly 
readily.  He  also  found  that  a  pressure  of  three  to  five  atmospheres 

1  Ztschr.  f.  Thiermedizin,  1897.  i,  241  2  'S. 

2  Ccntralbl.  f.  Bakt.,  Orig.,  1903,  xxxiii.  LJO 

3  Jour.  Med.  Research,  1912,  xxvi,  441. 


BACILLUS  ABORTUS  383 

would  facilitate  the  growth  of  the  organism.  On  dextrose  agar  the 
colonies  are  round,  normally  colorless  and  transparent,  and  have  a 
very  glistening,  pearly  sheen.  The  colonies  attain  a  diameter  of  from 
0.5  to  2.5  mm.  The  organism  grows  well  on  blood  serum.  On  gelatin 
the  growth  is  usually  very  slow,  probably  because  of  the  lowered  tem- 
perature of  incubation.  No  liquefaction  takes  place.  In  milk  there 
is  a  moderate  growth;  no  acid  is  formed,  and  no  coagulation  or  pep- 
tonization  takes  place. 

Conditions  of  Growth. — The  organism  is  killed  by  an  exposure  of  59° 
C.  for  ten  minutes.1 

Products  of  Growth. — The  organism  produces  no  known  ferments 
and  it  produces  no  acid  in  dextrose  or  other  sugars;  on  the  contrary, 
the  reaction  on  artificial  media  in  which  the  organism  is  growing 
becomes  slightly  alkaline.  The  organism  forms  no  extracellular  toxins. 

Pathogenesis. — Infectious  abortion  appears  to  be  an  infection  of  the 
fetus  in  utero  and  its  membranes,  which  results  in  the  death  of  the 
fetus  and  its  expulsion,  or  less  commonly  its  expulsion  in  a  living 
and  enfeebled  state.  The  time  of  expulsion  is  not  definite;  it  may 
occur  early  during  the  period  of  gestation,  or  it  may  not  take  place 
until  the  normal  completion  of  pregnancy.  Ordinarily  there  is  no 
direct  evidence  of  disease  in  the  mother. 

The  lesions  in  experimental  guinea-pigs,  which  have  been  described 
very  carefully  by  Fabyan,2  resemble  both  macroscopically  and  his- 
tologically  those  of  tuberculosis.  As  a  rule,  the  "muscles  are  free  from 
lesions  and  there  is  a  tendency  for  the  organism  to  localize  itself  in 
the  perivascular  or  subcapsular  regions  of  various  abdominal  organs. 
The  organism  may  persist  in  experimental  animals  for  very  consider- 
able periods  of  time  without  producing  manifest  symptoms.  Fabyan 
has  shown3  that  the  organism  may  remain  alive  but  latent  in  guinea- 
pigs  for  over  a  year. 

Immunity. — Cows  which  have  aborted  once  or  twice  appear  to  acquire 
an  immunity  which  is  supposed  to  be  due  to  the  formation  of  anti- 
bodies in  the  blood.  Although  no  extracellular  toxins  have  been 
demonstrated  as  yet,  it  is  probable  that  the  infected  animal  is  sensitized 
by  endotoxins  of  the  abortion  bacillus,  for  such  animals  injected  with 
"Abortin"  (an  extract  of  the  abortus  bacillus)  usually  give  a  definite 
reaction. 

Of  extreme  importance  is  the  frequent  occurrence  of  the  organism 

1  Fabyan,  loc.  cit.,  p.  481.  2  Loc.  cit. 

3  Jour.  Med.  Research,  1913,  xxviii,  81. 


384  GLANDERS— ANTHRAX— PYOCYANEUS 

in  milk.  Melvin1  has  found  B.  abortus  in  eight  out  of  seventy-seven 
samples  of  market  milk  and  in  the  milk  of  six  dairies  out  of  a  total  of 
thirty-one  examined.  As  early  as  1894  Theobald  Smith2  called  atten- 
tion to  peculiar  tubercle-like  lesions  induced  in  guinea-pigs  following 
the  injection  of  cow's  milk.  He  recognized  that  the  disease  was  not 
tuberculosis;  later  Schroeder3  made  similar  observations.  In  the 
same  year  Smith  and  Fabyan4  showed  that,  the  tubercle-like  lesions 
were  caused  by  B.  abortus,  and  in  1913  Fabyan5  demonstrated  con- 
clusively the  extremely  important  fact  that  B.  abortus  is  very  fre- 
quently found  in  the  milk  of  cows  that  have  aborted.  He  also  showed 
that  pasteurization  of  milk,  if  carried  out  in  the  proper  manner,  will 
certainly  destroy  the  bacillus.  Whether  certain  cases  of  abortion 
observed  in  man  are  due  to  the  organism  is  not  yet  proven.6 

Bacteriological  Diagnosis. — The  bacteriological  diagnosis  is  best 
made  by  injecting  guinea-pigs  with  suspected  milk  or  material  from 
a  diseased  animal  and  observing  the  development  of  the  characteristic 
tubercle-like  lesions.  If  the  animal  does  not  die  within  a  reasonable 
time  it  should  be  killed  and  autopsied. 

Serological  Diagnosis. — The  blood  serum  of  infected  cattle  usually 
agglutinates  B.  abortus  in  dilutions  greater  than  1  to  50.  The  value 
of  the  agglutination  reaction  as  a  method  of  diagnosis  is  as  yet  debat- 
able. The  extensive  statistics  of  MacFadyen  and  Stockmann7  upon 
this  phase  of  the  subject  are  representative.  An  agglutination  with 
B.  abortus  in  a  dilution  of  1  to  50  was  obtained  with  the  sera  of  526 
out  of  a  total  of  535  apparently  healthy  cows;* in  the  remainder  (9) 
agglutination  took  place  in  dilutions  greater  than  1  to  50.  Of  127 
cattle,  either  infected  or  suspects,  an  agglutination  was  not  obtained 
in  a  dilution  of  1  to  50;  in  11  agglutination  was  positive,  1  to  50;  in 

19  a  positive  reaction  was  obtained  in  a  dilution  of  1  to  100;  and  in 

20  a  reaction  in  a  dilution  of  1  to  200.    Holth8  tested  the  sera  of  7 
normal  cattle  with  negative  results.    The  sera  of  38  animals  out  of  a 
total  of  39,  which  were  plainly  infected  with  B.  abortus,  gave  positive 
agglutination  with  the  specific  organism  in  a  dilution  of  1  to  100. 

1  Vet.  Jour.,  1912,  Ixviii,  526. 

2  Bureau  of  Animal  Industry,  1894,  Bull.  7,  80. 

3  Bur.  Animal  Industry,  Circ.  198,  November  2,  1912. 
4Centralbl.  f.  Bakt.,  Orig.,  1912,  Ixi,  549. 

6  Jour.  Med.  Research,  1913,  xxviii,  85. 

6  Recently  Laisen  and  Sedgwick  (Am.  Jour.  Dis.  of  Child.,  1913,  vi,  326)  have  exam- 
ined blood  serum  from  425  children  by  the  method  of  complement-fixation;  73  were 
positive,  325  weie  negative. 

7  Jour.  Compt.  Path,  and  Therap.,  1912,  xxv,  22. 

8  Berl.  tierarztl.  Wchnschr..  1909,  686. 


ACIDURIC  BACTERIA  385 

The  method  of  complement  fixation,  precipitin  test,  ophthalmo  reac- 
tion and  intracutaneous  reaction  with  various  preparations  of  B. 
abortus  have  been  tested  for  their  diagnostic  value,  but  the  results 
are  not  clear  cut  and  definite.1 

Prophylaxis  and  Dissemination. — The  infection  of  market  milk  with 
B.  abortus  focuses  attention  sharply  upon  the  transmissibility  of  the 
organism  to  man.  Definite  details  are  lacking,  but  pasteurization  of 
milk  should  remove  all  practical  danger  from  this  source. 

ACIDURIC   BACTERIA.2 

There  is  a  somewhat  poorly  defined  group  of  bacilli,  chiefly  found 
in  the  intestinal  contents  of  man  and  animals,3  which  possesses  the 
unusual  property  of  growing  in  fermentation  media  of  a  degree  of 
acidity  incompatible  with  the  development  of  all  other  known  bacteria. 
The  aciduric  bacteria  are  of  two  kinds :  the  true  acidurjc  bacilli,  of 
which  Bacillus  acidophilus  is  the  best  known,  and  facultatively 
aciduric  bacteria,4  which  are  occasionally  detected  in  the  intestinal 
contents  of  man  and  animals  fed  for  some  time  upon  carbohydrate. 
The  facultative  organisms  rapidly  lose  their  acid  tolerance  upon  cul- 
tivation in  ordinary  media,  and  they  are  probably  to  be  regarded  as 
examples  of  bacterial  adaptation. 

Rahe5  distinguishes  three  types  of  aciduric  bacilli,  depending  upon 
their  action  upon  carbohydrates.  Acid,  but  no  gas  is  formed,  as 
follows : 

Type  I.  Bacillus  bulgaricus  (not  an  intestinal  organism)  coagulates 
m  ilk,  but  does  not  ferment  mannite. 

Type  II.  Coagulates  milk  and  ferments  mannite. 

Type  III.  Does  not  coagulate  milk,  but  ferments  mannite. 

Bacillus  Acidophilus. — Bacillus  acidophilus,  described  by  Moro6 
and  independently  by  Finkelstein7  is  a  somewhat  pleiomorphic  bacillus 
of  varying  length,  which  occurs  singly  or  in  pairs  as  a  rule.  Chain 
formation  is  not  uncommonly  observed  in  cultures  on  artificial  media. 
The  organism  forms  no  spores  or  capsules  and  it  is  typically  Gram- 

1  See  Klimmer,  Ergebnisse  der  Immunitatsforsch.  u.  experimentelle  Therap.,   1914,  i, 
143-188,  for  details. 

2  Kendall,  Jour.  Med.  Research,  1910,  xxii,  153 — for  resume  and  literature  to  1910. 
See  also  Rahe,  Jour.  Inf.  Dis.,  1914,  xv,  141. 

3  Mereschkowsky,  Centralbl.  f.  Bakt.,  Orig.,  1905,  xxxix,  380,  584,  696;    1906,  xl,  118. 

4  Kendall,  loc.  cit.,  p.  165. 

5  Loc.  cit. 

6  Wien.  klin.  Wchnschr.,  1900,  v,  114. 

7  Deutsch.  med.  Wchnschr.,  1900,  xxii,  263. 

25 


386  GLANDERS— ANTHRAX— PYOCYANEUS 

positive,  although  in  old  cultures  a  majority  of  the  bacilli  are  fre- 
quently Gram-negative. 

Isolation  and  Culture. — The  organism  may  be  isolated  directly  from 
suspected  material  in  2  per  cent,  dextrose  broth  containing  0.25  per 
cent,  acetic  acid.  After  two  or  three  days  growth  becomes  apparent 
and  a  few  loopfuls  of  the  well-shaken  culture  are  transferred  to  a  second 
and  then  a  third  tube  of  the  same  medium.  Usually  the  third  transfer 
contains  either  a  pure  culture  or  it  is  greatly  enriched  with  the  specific 
organism.  Pure  colonies  are  obtained  by  plating  upon  2  per  cent, 
dextrose  agar  unadjusted  for  reaction,  or  better,  upon  dextrose  agar 
containing  0.2  per  cent,  sodium  oleate  according  to  the  procedure  of 
Salge.1 

The  colonies  are  of  two  types — a  round,  smooth-edged  compact 
colony,  and  a  thin,  semi-translucent  colony  with  delicate  filamentous 
edges. 

Products  of  Growth. — Bacillus  acidophilus  is  carbohydrophilic  in  its 
activities;  it  does  not  grow  well  in  media  containing  proteins  and 
protein  derivatives  only.  Indol,  phenols  and  similar  products  of 
protein  degradation  are  not  found  in  cultures  of  this  organism.  Gelatin 
is  not  liquefied  and  growth  is  feeble  in  this  medium. 

Frequently  cultures  on  sodium  oleate  agar  slants  exhibit  a  clouding 
of  the  medium;2  the  cause  of  the  clouding  is  not  known. 

Pathogenesis. — Escherich3  and  Salge4  have  described  acute  diarrheas 
in  young  children,  characterized  bacteriologically  by  large  numbers 
of  Gram-positive  bacilli  in  the  feces  which  are  strongly  acid  in  reac- 
tion; Escherich  applied  the  name  "Blaue  Bazillose"  to  this  type  of 
intestinal  disturbance,  because  of  the  great  preponderance  of  Gram- 
positive  bacilli  in  Gram-stained  preparations  prepared  directly  from 
the  feces.  Subsequent  investigations  have  shown  that  the  ublue 
bacilli"  were  in  all  probability  Bacillus  acidophilus,  and  it  has  been 
shown  that  a  condition  apparently  identical  with  that  described  by 
Salge  may  develop  in  young  children  fed  with  too  much  maltose  or 
malz  suppe.5 

Bacillus  Acidophil-aerogenes. — Torrey  and  Rahe6  have  described  a 
member  of  the  aciduric  group  of  bacteria  which  produces  acid  and  gas 

1  Jahrb.  f.  Kinderheilk.,  1904,  lix,  399. 

2  Kendall,  loc.  cit.,  p.  156;  Rahe,  loc.  cit.,  p.  9. 

3  Jahrb.  f.  Kinderheilk.,  1900,  Hi,  1. 

4  Kie  akute  Diinndarmkatarrh  des  Sauglings,  Leipzig,  1906. 

5  Kendall,  Boston  Med.  and  Surg.  Jour.,  1910,  clxiii,  322. 

6  Jour,  of  Inf.  Dis.,  1915,  xvii,  437. 


ACIDURIC  BACTERIA  387 

in  dextrose,  lactose,  saccharose  and  maltose;  mannite  was  not  fermented. 
The  morphology  of  the  organism,  the  types  of  colonies  produced  on 
dextrose  agar,  and  its  staining  reactions  resemble  those  of  Bacillus 
acidophilus.  The  production  of  gas  in  the  sugar  mentioned  and 
the  relatively  feeble  growth  in  milk  are  its  distinguishing  cultural 
characteristics. 

Sera  of  animals  immunized  to  Bacillus  acidophil-aerogenes  failed 
to  agglutinate  Bacillus  acidophilus,  and  vice  versa,  indicating  that  the 
two  organisms  are  quite  distinct  entities. 


CHAPTER   XX. 


THE  DIPHTHERIA  BACILLUS  GROUP. 


THE  DIPHTHERIA  BACILLUS. 

BACILLI   SIMILAR   TO  THE    DIPHTHERIA 


BACILLUS. 


Bacillus  Hofmanni. 
Bacillus  Xerosis. 
Bacillus  Hodgkini. 


THE   DIPHTHERIA   BACILLUS. 


Synonyms. — Corynebacterium  diphtherise,  Klebs-Loffler  bacillus. 

Historical. — A  small  group  of  bacteria  excrete  soluble  extracellular 
toxins  which  produce  specific  disease.  The  first  member  of  the 
group  to  be  isolated  and  studied  was  the  diphtheria  bacillus.  Klebs1 
called  attention  to  the  very  general  occurrence  of  a  bacillus  of  unusual 
and  characteristic  appearance  in  the  gray  membranes  usually  present 
in  the  throats  of  severe  and  fatal  cases  of  diphtheria,  and  a  year 
later  Loffler2  isolated  the  organism  in  pure  culture  from  several  cases 
of  the  disease.  Loffler  also  obtained  the  diphtheria  bacillus  from  the 
throat  of  an  apparently  normal  child,  which  led  him  to  be  very  guarded 
in  attributing  a  specific  relationship  of  the  organism  to  the  disease. 
Subsequent  studies  by  innumerable  investigators  have  corroborated 
these  observations  in  every  essential  detail,  and  have  demonstrated 
conclusively  that  the  diphtheria  bacillus  is  the  specific  etiological 
organism  of  diphtheria.  Roux  and  Yersin3  discovered  the  soluble 
toxin  of  the  diphtheria  bacillus  and  reproduced  the  essential  systemic 
phenomena  of  the  disease  in  experimental  animals  by  injecting  the 
toxin  freed  from  bacteria  by  filtration  through  porcelain.  V.  Behring 
and  Kitasato4  made  the  very  important  discovery  that  the  blood 
serum  of  animals  injected  with  gradually  increasing  amounts  of  diph- 
theria toxin  contained  a  specific  antitoxin  which  would  neutralize  the 
toxin;  diphtheria  antitoxin  is  one  of  the  very  few  specific  sera  possess- 
ing curative  properties. 

Morphology.— The  diphtheria  bacillus  is  one  of  the  very  few  bacteria 
which  possess  a  characteristic  morphology.  The  organisms  are 

1  Verhandl.  Kong.  Inn.  Med.,  Wiesbaden,  II.  Abt.,  1883,  143. 

2  Mitt.  a.  d.  kais.  Gesamte,  1884,  ii,  451. 

3  Ann.  Inst.  Past.,  1888,  642. 

4Deutsch.  med.  Wchnschr.,  1890,  xvi,  1113. 


THE  DIPHTHERIA  BACILLUS  389 

highly  pleiomorphic  bacilli,  usually  slender  straight  or  slightly  curved 
rods  with  rounded  and  frequently  swollen  ends.  The  size  and  shape 
of  the  individual  organisms  vary  greatly  even  in  the  same  culture; 
they  are  not  uniformly  cylindrical  as  a  rule,  but  have  club-like  thick- 
enings at  one  or  both  ends,  or  they  are  swollen  in  the  middle  and  more 
or  less  pointed  at  the  ends.  Occasionally  one  end  only  is  thickened, 
giving  rise  to  a  long,  somewhat  wedge-shaped  rod.  The  distinctive 
morphology  is  best  seen  in  eighteen-  to  twenty-four-hour  growths  on 
Loffler's  coagulated  blood  serum;  organisms  from  growths  on  agar 
are  more  uniform  in  appearance.  Diphtheria  bacilli  observed  directly 
in  diphtheritic  membranes  are  also  less  pleiomorphic  than  those  from 
blood  serum  cultures.  The  organisms  occur  singly  or  in  pairs,  very 
uncommonly  in  short  chains.  The  size  is  very  variable,  ranging  from 
0.3  to  0.8  micron  in  diameter  and  from  1.5  to  6  microns  in  length. 
The  organism  as  ordinarily  seen  in  diphtheritic  membranes  is  about 
0.6  micron  in  diameter  and  about  4  microns  in  length.  Branched 
forms  are  occasionally  seen,  particularly  in  the  membrane  which 
forms  on  old  plain  broth  cultures. 

The  stainable  substance  of  the  organisms  is  not  uniformly  dis- 
tributed, but  occurs  in  somewhat  irregular  concentration,  giving  rise 
to  three  rather  distinct  types  of  bacilli:  the  granular,  the  barred,  and 
the  solid.1  Metachromatic  granules  (Ernst-Babes  granules)  are  also 
present,  and,  according  to  Williams,  the  diphtheria  bacillus  reproduces 
by  fission  at  one  of  these  granules.  It  was  originally  supposed  that 
the  metachromatic  granules  were  only  found  in  virulent  strains  and 
that  the  non-virulent  strains  had  no  granules.  Neisser2  invented  a 
stain  which  brings  out  these  granules  very  sharply.3  It  is  now  known 
that  the  granules  are  not  necessarily  related  to  virulence,  conse- 
quently the  Neisser  stain  is  rarely  used.  Diphtheria  bacilli  stain 
well  with  the  ordinary  anilin  dyes  and  very  characteristically  with 

1  Wesbrook,  Wilson,  and  McDaniel,  Jour.  Boston  Soc.  Med.  Sc.,  1900,  iv,  75;    Trans. 
Assn.  Am.  Phys.,  1900. 

2  Ztschr.  f.  Hyg.,  1897,  xxiv,  443. 

3  The  stain  is  prepared  in  the  following  manner: 

A. — Methylene-blue  (Griibler's) 1  gram 

Alcohol,  96  per  cent 20  c.c. 

Glacial  acetic  acid 50  c.c. 

Distilled  water 950  c.c. 

B. — Bismarck  brown .          1  gram 

Distilled  water  .      . 500  c.c. 

The  smear,  fixed  in  the  flame  in  the  usual  manner,  is  covered  with  solution  A  for  three 
to  five  seconds,  washed  in  water,  then  covered  with  B  for  three  to  five  seconds.  After 
thorough  washing  in  water,  the  preparation  is  ready  for  microscopic  examination. 
The  granules  are  stained  blue,  the  bodies  of  the  bacilli  brown. 


390 


THE  DIPHTHERIA   BACILLUS  GROUP 


Loffler's  methylene  blue.  With  methylene  blue  the  granules  above 
mentioned  are  brought  out  very  sharply,  and  it  is  observed  that  these 
granules  exhibit  the  phenomenon  known  as  metachromatism,  that  is, 
they  stain  mahogany  red  while  the  rest  of  the  organism  stains  blue. 
Diphtheria  bacilli  are  Gram-positive,  but  prolonged  washing  with 
alcohol  removes  the  Gram-positive  stain.  Cultures  prepared  directly 
from  diphtheritic  membranes  stain  more  uniformly  than  organisms 
obtained  from  cultures  on  Loffler's  blood  serum. 

Diphtheria  bacilli  are  non-motile,  possess  no  capsules  and  form  no 
spores.  Very  frequently  the  organisms  are  arranged  in  a  definite 
and  characteristic  manner,  occurring  very  commonly  in  pairs,  each 
pair  of  organisms  forming  a  configuration  very  similar  to  a  capital 
"L,"  and  a  series  of  these  angulated  pairs  are  arranged  in  parallel, 


FIG.  55.— Bacillus  diphtherias,  methylene-blue  stain.    (  X  1000.) 


very  much  like  chevrons.    This  angular  arrangement  of  the  organisms 
is  due  to  their  method  of  reproduction. 

Isolation  and  Culture. — The  diphtheria  bacillus  grows  best  on  Loffler's 
alkaline  blood  serum  and  this  medium  is  almost  specific  for  the 
organism,  which  during  the  first  nine  to  eighteen  hours'  incubation 
outgrows  all  other  organisms  with  which  it  is  usually  associated  in 
characteristic  lesions,  except  staphylococci.  Colonies  of  diphtheria 
bacilli  on  this  medium  after  eighteen  hours'  incubation  at  37°  C.  are 
gray-white,  round,  rather  dull,  with  darker  centres,  and  may  attain 
a  diameter  of  1  to  1.5  mm.  Diphtheria  bacilli  grow  somewhat  more 
slowly  on  plain  agar,  forming  small,  non-characteristic  colonies.  The 
organisms  produce  a  well-marked  zone  of  hemolysis  around  the 
individual  colonies  on  blood  agar,  but  the  hemolytic  area  is  smaller 


THE  DIPHTHERIA  BACILLUS  391 

than  that  characteristic  of  the  streptococcus.  Pseudodiphtheria  bacilli 
do  not  produce  hemolysis  on  this  medium1  The  growth  of  diphtheria 
bacilli  in  gelatin  is  slow,  and  the  organisms  do  not  produce  liquefaction 
of  the  medium.  In  plain  broth  the  organism  grows  rather  slowly; 
repeated  transfers  are  usually  followed  by  the  development  of  a  pel- 
licle which  floats  on  the  surface.2  This  pellicle  may  sink,  but  a  new 
one  usually  takes  its  place.  The  growth  in  dextrose  broth  is  more 
luxuriant  than  in  plain  broth,  but  no  pellicle  forms.  There  is,  however, 
a  well-marked  turbidity.  The  diphtheria  bacillus  grows  well  in  milk, 
producing  an  initial  acid  reaction  during  the  first  two  or  three  days 


FIG.  56.— Bacillus  diphtherias,  branching.     ( X  800.) 

of  incubation,  followed  by  the  gradual  development  of  an  alkaline 
reaction.3  No  gross  changes,  however,  are  produced  in  the  milk,  even 
with  prolonged  cultivation.  The  growth  on  potato  is  very  slight 
provided  the  reaction  of  the  potato  is  alkaline;  no  growth  at  all 
takes  place  on  acid  potato. 

The  diphtheria  bacillus  is  an  aerobic,  facultatively  anaerobic  organ- 
ism. Its  limits  of  growth  are  17°  C.  as  a  minimum,  43°  C.  as  a  maxi- 
mum, with  the  optimum  at  37°  C.  Ten  minutes  exposure  to  60° 
C.,  five  minutes  at  70°  C.,  or  one  minute  at  100°  C.  readily  kills  diph- 
theria bacilli.  The  organisms  are  occasionally  transmissible  through 
milk,  and  in  this  connection  it  should  be  remembered  that  the  ordinary 
method  of  heating  milk  in  an  open  vessel  will  not  certainly  kill  diph- 

1  Mandelbaum  and  Heinemann,   Centralbl.  f.  Bakt.,  Orig.,  1910,  Hii,  356;    Rankin, 
Jour.  Hyg.,  1911,  xi,  271. 

2  It  is  essential  for  the  production  of  toxin  that  the  organisms  be  cultivated  until 
they  produce  a  pellicle,  leaving  the  underlying  medium  perfectly  clear  and  free  from 
bacilli. 

3  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Assn.,  1914,  xxxvi,  195. 


392  THE  DIPHTHERIA   BACILLUS  GROUP 

theria  bacilli,  for,  as  Theobald  Smith1  pointed  out  many  years  ago, 
a  scum  forms  on  the  free  surface  of  the  milk,  consisting  of  casein  and 
lime  salts,  which  is  a  non-conductor  of  heat.  Within  this  membrane 
the  diphtheria  bacilli  may  resist  a  long  period  of  heating.  Diphtheria 
bacilli  exposed  to  heat  enclosed  in  a  false  membrane,  as  for  example, 
those  taken  from  the  throat,  may  resist  an  exposure  of  95°  to  100°  C. 
even  for  an  hour.  Organisms  dried  in  this  membrane  may  remain 
viable  at  low  temperatures,  protected  from  sunlight,  from  three  to 
five  months.  Naked  germs  are  readily  killed  by  antiseptics  in  the 
ordinary  concentrations,  but  those  exposed  to  the  action  of  antiseptics 
protected  in  membranes  may  resist  for  some  time.  Hydrogen  peroxide 
is  said  to  be  particularly  germicidal  for  the  diphtheria  bacillus. 

Products  of  Growth. — Chemical. — Bacillus  diphtheria  produces  acids, 
chiefly  lactic,  together  with  smaller  amounts  of  formic  acid  from  the 
fermentation  of  dextrose  and  maltose.  Lactose,  saccharose  and 
mannite  are  not  fermented.  Neither  indol  nor  phenols  are  formed 
in  sugar-free  broths,2  but  small  amounts  of  ammonia  are  produced 
in  this  medium,  the  amount  increasing  with  the  age  of  the  culture.3 

Enzymes. — No  enzymes  acting  upon  proteins,  carbohydrates  or 
fats  have  been  detected  in  cultures  of  the  organism. 

Toxin. — The  most  important  and  characteristic  of  the  products 
formed  by  the  diphtheria  bacillus  is  a  potent,  soluble  (extracellular) 
toxin.  The  potency  of  the  toxin  varies  somewhat  with  the  culture 
used,  some  strains  producing  more  than  others.  An  occasional  strain, 
typical  in  other  respects,  fails  to  form  toxin.  Prolonged  cultivation 
of  the  organism  in  artificial  media  may  lead  to  a  diminution  in  toxin- 
producing  capacity,  but  this  is  by  no  means  a  general  rule.  Williams4 
isolated  a  diphtheria  bacillus  from  a  mild  case  of  tonsillar  diphtheria 
(No.  8)  which  has  retained  its  toxin-producing  power  unimpaired  up 
to  the  present  time.  This  culture  is  widely  used  throughout  the  world 
in  the  commercial  preparation  of  diphtheria  antitoxin. 

Conditions  Favoring  the  Production  of  Diphtheria  Toxin. — 1.  Com- 
position of  the  Medium. — Park  and  Williams5  found  that  2  per  cent, 
of  peptone  added  to  meat  infusion  broth  increased  the  yield  of  toxin 
very  materially,  and  Theobald  Smith6  made  the  very  important 

1  Theobald  Smith,  Jour.  Exp.  Med..  1899,  iv,  233. 

2  Ibid.,  1897,  ii,  543. 

3  Kendall,  Day  and  Walker,  Jour.  Am.  Chem.  Soc..  1913.  xxxv,  1210. 

4  Jour.  Med.  Research,  June,  1902. 
6  Jour.  Exp.  Med.,  1896,  i,  164. 

«Tr.  Assn.  Am,.  Phys.,  1896:   Jour.  Exp.  Med.,  1899,  iv,  373. 


THE  DIPHTHERIA   BACILLUS  393 

observation  that  the  presence  of  muscle-sugar  (dextrose),  commonly 
found  in  small  amounts  in  meat-juice,  prevented  the  formation  of 
diphtheria  toxin;  he  demonstrated  conclusively  that  small  amounts 
of  dextrose  (less  than  0.2  per  cent.)  delay  the  appearance  of  toxin; 
in  sugar-free  broth  toxin  production  increases  with  the  growth  of  the 
organisms.  Diphtheria  toxin  is  formed  from  the  protein  constituents 
of  the  medium;  when  utilizable  carbohydrate  (dextrose)  is  present 
in  the  medium,  the  bacilli  ferment  it  instead  of  attacking  the  protein.1 
It  is  customary  to  add  0.1  per  cent,  of  dextrose  to  broth  for  the  pro- 
duction of  diphtheria  toxin;  the  initial  development  of  the  bacilli  is 
greater,  and  this  amount  of  dextrose  is  rapidly  used  up,  leaving  greater 
numbers  of  organisms  to  form  toxin  from  the  protein  constituents. 
The  culture  must  be  grown  at  37°  C.  to  insure  a  potent  toxin. 

2.  Oxygen. — Free  oxygen  is  an  essential  factor  in  the  production 
of  toxin.     It  is  customary  to  distribute  the  broth  in  shallow  layers 
with  a  relatively  large  surface  exposed  to  the  air. 

3.  Pellicle. — Cultures  of  diphtheria  bacilli  which  grow  habitually 
on  the  surface  of  fluid  media  must  be  used  for  the  preparation  of 
toxin.    Diphtheria  bacilli  can  be  "trained"  to  develop  on  the  surface 
by   repeated   transfers   in   broth.2     Surface   development    insures  a 
maximal  exposure  of  the  bacilli  to  the  air. 

4.  Incubation. — It  requires  from  seven  to  ten  days'  incubation  at 
37°  C.  for  the  maximum  accumulation  of  toxin.    Deterioration  of  the 
toxin  after  this  time  sets  in,  and  the  formati6h  of  new  toxin  fails  to 
keep  pace  with  the  recession  in  potency  of  the  toxin  already  formed.3 

Storage  of  Toxin. — At  the  end  of  the  period  of  incubation  carbolic 
acid  or  other  preservative  is  added  to  the  broth  to  kill  the  bacilli; 
they  rapidly  settle  out,  leaving  a  clear  supernatant  fluid  free  from 
bacteria,  containing  the  toxin,  which  is  either  decanted  off  from  the 
bacilli  or  filtered  through  unglazed  porcelain  to  remove  the  bacteria. 
It  is  then  stored  in  amber  bottles  which  are  completely  filled  and 
kept  in  cold  storage.  Under  these  conditions  the  toxin  deteriorates 
comparatively  slowly. 

Testing  Toxin. — Toxin  produced  by  the  diphtheria  bacillus  kills 
the  ordinary  laboratory  animals,  guinea-pigs,  rabbits,  dogs,  and  birds; 
but  it  is  practically  without  effect  upon  rats  and  mice,  unless  the 
toxin  is  injected  directly  into  the  nervous  system.  The  general  method 

1  Kendall,  Boston  Med.  and  Surg.  Jour.,  1913,  clxviii,  825. 

2  Theobald  Smith,  Jour.  Exp.  Med.,  1899,  iv,  392. 

3  Ibid. 


394  THE  DIPHTHERIA  BACILLUS  GROUP 

of  testing  the  potency  of  the  toxin  is  to  inject  successively  smaller 
graduated  doses  of  it  subcutaneously  into  guinea-pigs  of  two  hundred 
and  fifty  grams  weight  and  observe  the  results.  The  smallest  amount 
of  a  toxin  which  will  kill  a  guinea-pig  weighing  two  hundred  and  fifty 
grams  in  four  days  is  designated  the  minimal  lethal  dose  (M.  L.  D.). 
The  minimal  lethal  dose  varies  considerably  with  different  strains 
of  bacilli;  in  general  it  varies  from  0.25  c.c.  to  0.001  c.c.  The  injec- 
tion of  a  M.  L.  D.  of  toxin  leads  to  an  edematous  swelling  at  the  site 
of  inoculation  and  the  animal  soon  exhibits  generalized  symptoms  as 
well;  the  temperature  rises,  the  respirations  are  hurried,  and  death 
ensues  from  the  results  of  the  toxemia.  The  more  acute  the  death, 
the  less  striking  the  symptoms  and  lesions.  Guinea-pigs  which  have 
died  on  the  fourth  day  exhibit  a  marked  congestion  of  the  abdominal 
and  thoracic  viscera  and  of  the  colon.  A  hemorrhagic  infiltration  and 
enlargement  of  the  suprarenals  is  almost  pathognomonic.  Frequently 
the  stomach  wall  is  markedly  injected  with  blood  and  small  ulcera- 
tions  are  demonstrable  in  the  mucosa.1  The  lesions  present  the  same 
general  appearance  when  both  toxin  and  bacilli  are  injected,  but  a 
false  membrane,  composed  of  bacteria  and  a  fibrinopurulent  exudate, 
forms  at  the  site  of  inoculation.  The  bacilli  do  not  spread  to  other 
parts  of  the  body,  however,  but  remain  strictly  localized.  The  changes 
in  the  visceral  organs  are  attributable  to  the  absorption  of  the  toxin. 
A  sub-lethal  dose  of  toxin  or  an  attenuated  culture  of  diphtheria  bacill  i 
does  not  cause  death;  an  ulcer  forms  at  the  site  of  inoculation  which 
eventually  sloughs  away  and  is  completely  replaced  by  scar  tissue. 

Constitution  of  Diphtheria  Toxin. — The  composition  of  diphtheria 
toxin  is  unknown,  although  many  investigations  have  been  made  upon 
it.  Attempts  to  demonstrate  that  the  toxin  is  non-protein  in  nature 
by  growing  the  organisms  in  protein-free  media  have  not  been  con- 
vincing. Small  amounts  of  toxin  have  been  detected  in  these  cultures, 
but  the  well-recognized  synthetic  powers  of  bacteria  make  this  line 
of  evidence  inconclusive.  There  are  two  current  theories  which  receive 
serious  consideration.  One  theory  maintains  that  diphtheria  toxin  is 
enzymic  in  nature,  the  other  theory  assumes  that  the  toxin  is  related 
to  the  proteins,  particularly  the  globulins.  The  toxin  is  readily 
destroyed  by  exposure  to  light,  heat,  protoplasmic  poisons  and  to  peptic 
digestion,  consequently  moderate  amounts  of  it  may  be  swallowed 
without  apparent  harm.  Acids  destroy  the  toxin  slowly,  and  oxidizing 
agents,  as  hydrogen  peroxide,  iodin  and  iodin  trichloride,  reduce  the 

1  Rosenau  and  Anderson,  Jour.  Inf.  Dis.,  1907,  iv,  1. 


THE  DIPHTHERIA   BACILLUS  305 

toxicity  very  materially.  An  exposure  to  60°  C.  for  ten  hours,  or  at 
70°  C.  for  two  hours,  attenuates  the  toxin,  and  it  is  rapidly  inactivated 
or  destroyed  at  100°  C. 

Protein  precipitants,  as  ammonium  sulphate  and  alcohol,  precipitate 
the  toxin  from  the  broth  in  an  insoluble  state  with  but  little  reduction 
in  potency.  The  precipitate,  after  dialysis  to  remove  the  salts,  is 
soluble  in  water.  A  further  reduction  in  volume  and  partial  puri- 
fication can  be  attained  by  evaporating  the  broth  to  one-tenth  its 
original  volume  in  vacuo  at  a  temperature  not  exceeding  25°  C.  (in 
the  dark),  precipitating  with  alcohol,  filtering  and  dissolving  in  water, 
and  again  precipitating,  then  drying  the  precipitate  in  vacuo. 

Physiological  Action. — The  chemical  constitution  of  the  toxin  mole- 
cule is  unknown,  and  toxin  can  not  be  detected  or  assayed  chemically. 
It  provokes,  however,  a  definite  physiological  response  in  susceptible 
animals,  as  guinea-pigs,  and  its  presence  is  detected  and  its  strength 
determined  by  injecting  graduated  doses  into  them,  as  mentioned  above. 
From  its  physiological  action  the  toxin  molecule  appears  to  consist 
of  three  components  in  varying  amounts:  (a)  Toxin,  which  causes 
the  acute  symptoms  of  intoxication,  parenchymatous  degeneration 
and  death  when  injected  into  susceptible  animals.  This  fraction  of 
the  toxin,  according  to  Ehrlich,  has  a  special  affinity  for  the  antitoxin. 
(b)  Toxone:  the  toxone  causes  edema  at  the  site  of  inoculation  and 
the  postdiphtheritic  paralyses.  It  combines  with  antitoxin  more 
slowly  than  the  toxin,  (c)  Toxoid:  diphtheria  toxin  rather  readily 
loses  its  toxic  properties  on  standing,  retaining  its  power  of  com- 
bining with  antitoxin  unimpaired,  however.  Toxin  which  is  devoid 
of  toxic  power  but  which  combines  with  antitoxin  is  called  "toxoid." 

Antitoxin. — Preparation. — The  injection  of  the  soluble  toxin  of  the 
diphtheria  bacillus  in  sublethal  doses  into  experimental  animals  stimu- 
lates the  formation  of  specific  antitoxin  which  has  both  curative  and 
prophylactic  value.  Antitoxin  is  obtained  from  horses  because  they 
are  less  susceptible  to  the  action  of  the  toxin  than  smaller  animals. 
The  serum  of  horses,  at  least  in  single  doses,  is  innocuous  for  man,  and 
horses  furnish  large  amounts  of  blood  (containing  antitoxin)  without 
injury  to  the  animal.  Young  animals  free  from  glanders,  tuberculosis 
and  other  diseases  are  used  for  the  purpose.  Several  methods  are 
available  for  immunization,  but  the  one  commonly  selected  is  carried 
out  in  the  following  manner:  an  initial  injection  of  diphtheria  toxin, 
either  mixed  with  an  excess  of  antitoxin  or  attenuated  by  iodin 
trichloride,  is  made  and  about  a  week  later  a  second  injection  con- 


396  THE  DIPHTHERIA  BACILLUS  GROUP 

taining  an  increased  amount  of  toxin  follows.  At  regular  intervals 
the  injections  are  repeated,  each  time  increasing  the  amount  of  toxin 
in  regular  progression  until  after  three  to  four  months  as  much  as 
250  to  300  c.c.  of  unaltered  toxin  is  introduced  at  one  time.  After 
about  two  weeks  following  the  last  injection  the  animal  is  bled  and 
the  potency  of  the  serum  tested.  If  it  contains  one  hundred  and  fifty 
units  or  more  of  antitoxin  to  the  cubic  centimeter,  from  two  to  five 
liters  of  blood  are  removed  from  the  jugular  vein  with  sterile  precau- 
tions into  sterile  receptacles,  and  the  animal  is  again  treated  with 
toxin  to  induce  further  immunization.  As  a  rule,  about  two-thirds 
of  the  volume  of  blood  taken  is  regained  in  antitoxin-containing  serum. 
It  is  customary  in  large  establishments  to  immunize  several  horses 
at  the  same  time  and  mix  this  serum,  for  experience  has  shown  that 
the  serum  of  certain  animals  contains  substances  which  cause  erythe- 
matous  rashes  in  man  which  are  disagreeable  and  irritating  although 
not  necessarily  harmful.  Pooling  the  blood  reduces  this  possibility. 
The  serum  is  stored  in  sterile  containers  in  a  dark  cold  place  and 
retains  its  antitoxic  properties  well.  It  deteriorates  less  rapidly  than 
toxin. 

Concentration. — Atkinson1  noticed  that  the  globulins  of  the  horse 
serum  increased  and  the  albumins  diminished  as  the  antitoxin  content 
of  the  blood  increased,  and  he  effected  a  partial  purification  of  the  anti- 
toxin fraction  by  removing  the  albumin  with  protein  precipitants . 
Gibson2  carried  the  process  further  and  obtained  a  serum  which  was 
about  three  times  as  rich  in  antitoxin  per  unit  volume  as  the  original 
horse  serum.  Banzhaf3  has  reduced  the  proportion  of  non-specific 
protein  as  far  as  is  practical  by  purely  physical  agents.  This  reduction 
of  non-specific  proteins  is  important  for  two  reasons:  first,  because 
it  reduces  the  danger  of  anaphylaxis  due  to  sensitization  of  the  patient; 
and,  secondly,  because  the  rashes  and  joint  swellings  are  notably 
reduced  when  the  concentrated  antitoxin  is  used  instead  of  the  whole 
horse-serum.  It  is  possible  to  obtain  the  same  therapeutic  effect  by 
the  injection  of  about  one-third  the  amount  of  solution  when  con- 
centrated antitoxin  is  administered. 

Properties. — Diphtheria  antitoxin  specifically  neutralizes  diphtheria 
toxin  both  in  vitro  and  in  vivo.  It  has  little  neutralizing  value  for 
the  toxone,  however ;  consequently  in  severe  cases  when  it  is  used  late, 

1  Jour.  Exp.  Med.,  1899,  iii,  649. 

2  Jour.  Biol.  Chem.,  1906,  i,  Nos.  2  and  3. 

.3  Collected  studies  from  the  Research  Laboratory,  New  York  City  Board  of  Health, 
vols.  v  and  vi. 


THE  DIPHTHERIA  BACILLUS  397 

it  will  not  prevent  the  development  of  postdiphtheritic  paralyses.  It 
has  both  prophylactic  and  curative  properties.  It  is  not  bacteriolytic 
and  exhibits  no  agglutinins  for  diphtheria  bacilli.  Nothing  is  definitely 
known  of  the  nature  of  diphtheria  antitoxin.  If  the  diphtheria  toxin 
is  a  ferment,  the  antitoxin  would  appear  to  be  an  antiferment.  The 
fact  that  it  is  precipitated  with  the  globulin  fraction  of  the  blood  serum 
would  suggest  that  it  may  be  either  closely  related  to  the  proteins  or 
a  true  protein  itself. 

Diphtheria  toxin  varies  considerably  in  its  potency  due  to  the  fact 
that  it  deteriorates;  the  antitoxin,  on  the  contrary,  is  more  stable. 
Consequently  for  purposes  of  comparison  and  standardization  a 
standard  antitoxin  is  used.  Two  such  standard  antitoxins  are 
recognized  officially:  one  prepared  by  Ehrlich  in  Germany;  the  other 
prepared  by  the  United  States  Public  Health  Laboratory  in  Washing- 
ton, D.  C.  Both  of  these  antitoxins  were  prepared  on  a  very  large 
scale  and  preserved  in  a  cold,  dark,  dry  place  in  packages  of  conveni- 
ent size.  When  the  supply  of  one  or  the  other  of  the  standards  is 
nearly  exhausted  a  new  lot  of  antitoxin  will  be  prepared  and  carefully 
compared  with  the  old.  Small  amounts  of  the  standard  antitoxin 
containing  a  definite  number  of  antitoxin  units  are  sent  out  regularly 
by  the  central  laboratories  to  interested  laboratories  for  testing 
purposes. 

Standardization  of  Antitoxin. — The  antitoxin  unit  may  be  defined 
as  "that  amount  of  antitoxin  which  just  suffices  to  protect  a  guinea- 
pig  of  250  grams  weight  against  100  times  the  minimal  fatal  dose  of 
diphtheria  toxin."  The  process  of  standardization  of  antitoxin  of 
unknown  potency  is  carried  out  in  the  following  manner:  diphtheria 
toxin,  prepared  as  described  above,  is  mixed  in  gradually  diminishing 
amounts  with  a  definite  amount  of  the  standard  antitoxin  (containing 
a  known  number  of  antitoxic  units)  and  allowed  to  stand  for  twenty 
to  thirty  minutes  to  permit  union  of  the  toxin-antitoxin  to  take  place. 
The  mixtures  are  then  injected  subcutaneously  into  guinea-pigs  of 
250  grams  weight.  The  greatest  dilution  of  toxin  which  kills  a  guinea- 
pig  in  four  days  is  said  to  be  the  L+  dose — that  amount  of  toxin 
which  will  neutralize  (say)  100  antitoxin  units  and  leave  an  excess  of 
toxin  just  sufficient  to  kill  the  animal.  Having  found  the  L+  dose 
of  toxin  (which  standardizes  its  toxicity  in  terms  of  standard  anti- 
toxin), the  same  process  is  repeated,  using  this  L+  dose  of  toxin  mixed 
with  gradually  diminishing  amounts  of  the  antitoxin  to  be  stan- 
dardized. That  dilution  of  antitoxin  of  unknown  potency  which  will 


398  THE  DIPHTHERIA   BACILLUS  GROUP 

neutralize  all  except  sufficient  toxin  to  kill  a  250  gram  guinea-pig 
in  four  days  contains  100  antitoxin  units  in  the  example  cited. 
Knowing  the  dilution  of  the  antitoxin,  it  is  a  simple  problem  to  deter- 
mine the  number  of  units  in  1  c.c.  A  good  antitoxic  serum  should 
contain  from  200  to  700  units  per  cubic  centimeter  of  the  unconcen- 
trated  product. 

Curative  Value  of  Diphtheria  Antitoxin. — Diphtheria  antitoxin 
should  be  used  as  early  as  possible  in  order  to  obtain  the  maximum 
curative  effect.  This  is  clearly  set  forth  in  the  following  table.1 

Day  of  Cures. 


tess. 
1    .                    .       . 

Treated. 

7 

Cured. 

7 

Died. 

o 

Per  cei 
100 

2 

.      .                 71 

69 

2 

97 

3  . 

30 

26 

4 

87 

4  
5  
6 

,      ,      .      .     39 
.     1      .      .     25 
,      .-                17 

30 
15 
9 

9 
10 

8 

77 
60 
47 

7-14 

,      .            .     41 

21 

20 

51 

Indefinite 

3 

2 

1 

Totals 233  179  54  77 

According  to  Donitz  and  others,  the  initial  dose  of  antitoxin  should 
be  large;  it  is  believed  that  with  large  doses  of  antitoxin  even  some 
of  the  toxin  attached  to  the  tissue  cells  can  be  neutralized.  For  this 
purpose  4000  units  is  a  minimal  initial  dose,  and  severe  or  desperate 
cases  are  given  10,000  to  100,000  units.  The  antitoxin  should  be 
repeated  the  following  day  if  necessary.  It  is  better  to  administer 
too  much  than  too  little  antitoxin.  Antitoxin  given  subcutaneously 
is  least  dangerous  so  far  as  danger  from  anaphylaxis  is  concerned 
but  the  absorption  is  slow.  Intramuscular  injections,  particularly 
in  the  gluteal  region,  are  said  to  be  more  efficient  curatively.  In  des- 
perate cases  intravenous  injections  of  antitoxin  (without  carbolic 
acid  as  a  preservative  if  possible)  are  indicated. 

Active  Immunization  with  Toxin- Antitoxin  Mixtures. — Following  a 
suggestion  of  Theobald  Smith,2  Von  Behring3  has  attempted  to  create 
active  immunity  to  diphtheria  in  man  by  subcutaneous  injections  of 
toxin-antitoxin  mixtures  which  are  neutral  or  but  slightly  toxic  for 
guinea-pigs.  The  few  observations  which  are  available  are  on  the 
whole  encouraging,  but  do  not  justify  a  formal  opinion  of  the  practical 
value  of  this  procedure. 

1  Quoted  from  Citron,  Immunity. 

2  Jour.  Med.  Research,  1907,  xvi,  359. 

3  Deutsch.  med.  Wchnschr.,  1913,  p.  873. 


THE  DIPHTHERIA   BACILLUS  399 

The  Schick  Reaction.— Available  evidence  indicates  that  immunity 
to  infection  with  the  diphtheria  bacillus  depends  largely  upon  the 
antitoxin  content  of  the  blood,  and  systematic  studies  of  the  anti- 
toxin content  of  the  blood  of  infants,  children  and  adults  by  Schick,1 
Park,  Zingher  and  Scrota,2  Park  and  Zingher,3  Kolmer  and  Moshage,4 
Bundesen,5  and  Moody6  indicate  that  a  large  percentage — nearly  80 
per  cent,  of  young  infants,  50  per  cent,  of  children,  and  nearly  90 
per  cent,  of  adults — exhibit  sufficient  antitoxin  to  protect  them  against 
the  disease.  The  demonstration  of  antitoxin  in  the  blood  has  been 
simplified  greatly  by  Schick,  and  modified  somewhat  by  Park.7  It 
is  made  in  the  following  manner:  an  amount  of  diphtheria  toxin 
equivalent  to  one-fiftieth  the  minimal  fatal  dose  for  a  guinea-pig  is 
made  up  to  a  volume  of  0.2  c.c.  in  sterile  salt  solution  and  is  injected 
subcutaneously,  or  preferably  intracutaneously,  in  the  flexor  surface 
of  the  forearm.  Immediately  the  skin  is  raised  somewhat  as  the  fluid 
enters  the  tissues.  The  reaction  elicited  depends  upon  the  antitoxin 
content  of  the  blood,  a  positive  reaction  indicating  that  antitoxin 
is  absent,  or  present  in  minimal  amounts  appears  within  twenty-four 
hours  as  a  circumscribed  area  of  redness  and  a  more  diffuse  area  of 
induration  measuring  from  one-half  an  inch  to  more  than  an  inch 
in  diameter.  The  maximum  reaction  appears  within  forty-eight  hours 
and  disappears  within  a  week.  The  blood  of  a  patient  reacting  in 
this  manner  contains  less  than  one-thirtieth  of  a  unit  of  antitoxin 
per  cubic  centimeter.  A  fainter  reaction  is  frequently  exhibited, 
which  is  interpreted  to  mean  that  the  antitoxin  content  of  the  blood 
lies  approximately  between  one-fortieth  and  one  twenty-fifth  of  an 
antitoxin  unit  per  cubic  centimeter.  If  the  antitoxin  content  is  at 
least  one-twentieth  of  a  unit  per  cubic  centimeter,  the  reaction  is 
negative;  only  a  slight  reaction  results  due  to  the  wound  itself. 

Practically,  it  has  been  found  that  individuals  giving  a  negative 
reaction  possess  sufficient  antitoxin  to  protect  them  from  infection; 
nurses,  doctors,  ward  orderlies,  and  patients  who  react  negatively  do 
not  need  to  be  immunized  with  antitoxin  if  they  have  been,  or  are, 
exposed  to  the  infection.  Persons  giving  a  mild  or  severe  reaction 
should  be  immunized  with  prophylactic  doses  of  antitoxin. 

1  Miinchen.  med.  Wchnschr.,  1913,  Ix,  2608. 

2  Arch.  Pediatrics,  July,  1914. 

3  Proc.  New  York  Path.  Soc.,  N.  S.  1914,  xiv,  151. 

4  Am.  Jour.  Dis.  Child.,  1915,  p.  189. 

5  Jour.  Am.  Med.  Assn.,  1915,  Ixiv,  p.  1203. 

6  Ibid.,  1915,  Ixiv,  p.  1206. 

7  Loc.  cit. 


400  THE  DIPHTHERIA   BACILLUS  GROUP 

Pathogenesis. — Experimental  Evidence  of  Pathogenesis. — Loffler1  ap- 
pears to  have  been  the  first  to  attempt  to  establish  the  etiological 
relationship  of  the  diphtheria  bacillus  to  the  disease.  He  succeeded 
in  producing  diphtheritic  membranes  on  the  mucous  surfaces  of 
animals  by  rubbing  cultures  on  the  previously  injured  surface.  Num- 
erous laboratory  accidents,  where  the  organisms  have  been  inadver- 
tently swallowed  with  the  subsequent  development  of  typical  clinical 
diphtheria,  complete  the  proof  of  the*  etiological  relationship  of  the 
organism  to  the  disease. 

Animal  Pathogenesis. — Laboratory  animals,  excepting  mice  and 
rats,  are  very  susceptible  to  the  diphtheria  toxin.  Guinea-pigs  are 
particularly  susceptible,  and  the  subcutaneous  injection  of  fatal  or 
nearly  fatal  doses  of  broth  cultures  is  followed  after  one  to  three  days 
by  the  appearance  at  the  site  of  inoculation  of  a  membrane,  edema, 
and  a  serosanguineous  exudate.  A  pleuritic'  and  frequently  a  peri- 
cardial  exudate  are  found  as  well.  There  is  hyperemia  of  the  abdom- 
inal organs  and  a  very  characteristic  swelling  and  hyperemia  of  the 
adrenals.  The  kidneys  are  also  usually  hyperemic.  Often  there  are 
ecchymoses  and  even  ulcers  in  the  gastric  mucosa.  No  bacilli  are 
found  in  the  internal  organs.  Intraperitoneal  injections  are  less  severe 
as  a  rule  than  subcutaneous  inoculations  of  the  same  dose.  There 
is  usually  some  peritoneal  effusion  which  frequently  contains  diph- 
theria bacilli.  Intratracheal  inoculation  after  mechanical  injury  is 
commonly  followed  by  the  appearance  of  a  false  membrane  and  the 
animal  dies  of  toxemia;2  intravaginal  injection  after  injury  of  the 
mucosa  frequently  leads  to  a  necrotic  inflammation  with  membrane 
formation.3  Repeated  applications  of  diphtheria  toxin  to  the  con- 
junctiva of  rabbits  cause  a  marked  conjunctivitis  with  membrane 
formation.4 

Human. — In  man  diphtheria  bacilli  are  usually  localized  in  the 
false  membranes,  chiefly  on  the  tonsils  or  pharynx,  and  these  mem- 
branes may  extend  to  the  nose,  larynx,  and  mouth.  The  organisms 
occasionally  invade  the  blood  stream.  They  may  even  extend  into 
the  lungs  causing  a  true  bronchial  pneumonia.  Occasionally  diph- 
theria bacilli  may  cause  rhinitis  fibrinosa  or  simple  rhinitis.5  They 
also  are  found  in  occasional  cases  of  vulvitis  gangrenosa  and  noma 

1  Loc.  cit. 

2  Fraenkel,  Deutsch.  med.  Wchnschr.,  1895,  176. 

3  Roux  and  Martin,  Ann.  Inst.  Past.,  1894,  p.  625. 
4Morax  and  Elmassian,  Ann.  Inst.  Past.,  1898,  p.  219. 
5  Neumann,  Centralbl.  f.  Bakt.,  1902,  xxxi,  33. 


THE  DIPHTHERIA   BACILLUS  401 

faciei.1  Rarely,  false  membranes  are  found  on  the  genitalia  or  in 
cutaneous  wounds,  in  the  latter  case  producing  a  true  wound  diph- 
theria.2 The  association  with  certain  other  organisms,  particularly  the 
streptococcus,  the  staphylococcus,  and  B.  coli,  appears  to  increase  the 
virulence  of  the  diphtheria  bacillus.3 

Diphtheria  is  a  generalized  toxemia  with  a  local  infection.  The 
bacilli  cause  coagulation  necrosis  of  the  superficial  cells,  and  an  inflam- 
matory membrane  consisting  of  a  serofibrinous  exudate  in  which 
fibrin  and  leukocytes  are  prominent,  together  with  epithelial  cells, 
pyogenic  cocci,  and  diphtheria  bacilli  in  the  deeper  layers  adjacent 
to  the  denuded  epithelium.  At  times  the  membrane  strips  off  without 
serious  injury  to  the  underlying  epithelium,  but  in  severe  cases  the 
membrane  tears  away,  leaving  a  bleeding  raw  surface. 

There  are  three  principal  types  of  diphtheria:  the  faucial,  laryngeal, 
and  tracheal.  The  incubation  period  is  from  two  days  to  a  week.  An 
important  sequela  is  the  postdiphtheritic  paralysis,  which  is  sup- 
posed to  be  caused  by  the  toxone  component  of  the  diphtheria  toxin. 
This  is  anatomically  a  toxic  neuritis  and  it  occurs  in  from  10  to  20 
per  cent,  of  all  cases  of  diphtheria  from  2  to  4  weeks  after  the  attack. 
There  is  no  apparent  relation  between  the  severity  of  the  attack  and 
the  paralysis.  The  pharynx  is  most  commonly  affected,  next  in  order 
the  eyes,  leading  to  strabismus  (ptosis).  In  a  smaller  number  of  cases 
the  heart  is  affected.  When  the  heart  is  affected  the  patients  not 
infrequently  drop  dead  as  the  result  of  cardiac  failure.  The  early 
Use  of  antitoxin  usually  prevents  or  greatly  modifies  the  development 
of  postdiphtheritic  paralysis. 

Bacteriological  Diagnosis. — The  principle  involved  in  the  bacterio- 
logical diagnosis  of  diphtheria  (and  the  diagnosis  can  only  be  definitely 
established  by  bacteriological  examination)  is  to  make  cultures  from 
the  suspected  lesions  on  Loffler's  alkaline  blood  serum,  to  incubate 
the  culture  from  twelve  to  eighteen  hours  at  37°  C.,  to  stain  the 
resulting  growth  with  Loffler's  methylene  blue,  and  to  diagnose  the 
organisms  by  their  characteristic  morphology. 

The  Technic  of  Inoculation. — Rub  a  sterile  swab  on  the  under  surface 
of  the  diphtheritic  membrane,  avoiding  extraneous  organisms  and 
avoiding  touching  the  tongue  or  other  parts  of  the  mouth.  Smear 
this  infected  swab  gently  over  the  surface  of  the  S3rum,  rotating  the 

1  Freymouth,  Deutsch.  med.  Wchnschr.,  1898,  No.  15. 

2  Schottmiiller,  Deutsch.  med.  Wchnschr.,  1895,  p.  273. 

3  Theobald  Smith,  Medical  Record,  May,  1896. 
26 


402  THE  DIPHTHERIA   BACILLUS  GROUP 

swab  while  doing  so  to  bring  every  part  of  it  in  contact  with  the 
medium.  The  serum  is  incubated  at  37°  C.  for  twelve  to  eighteen 
hours.  It  is  customary  in  many  laboratories  to  make  a  preliminary 
examination  of  the  growth  on  the  serum  after  five  hours'  incubation, 
and  also  to  make  a  smear  from  the  swab  itself  after  the  serum  has 
been  inoculated  with  it.  By  these  preliminary  examinations  from  30 
to  60  per  cent,  of  diagnoses  may  be  correctly  anticipated.  During  the 
first  eighteen  hours  of  incubation  diphtheria  bacilli  outgrow  practically 
all  other  organisms.  After  this  time  the  other  organisms  tend  to  out- 
grow the  diphtheria  bacillus. 

Results. — 1.  Negative. — Negative  results  may  be  due  to  several 
factors:  (a)  the  absence  of  diphtheria  bacilli;  (b)  lack  of  care  in 
taking  the  culture,  either  failure  to  touch  the  infected  membrane,  or 
making  preparations  immediately  after  the  use  of  antiseptic  gargles; 
(c)  improper  smears  and  improper  stains;  (d)  poor  media;  (e) 
improper  interpretation. 

2.  Positive. — Positive  results  do  not  necessarily  prove  that  the 
patient  has  diphtheria  for  carriers  of  diphtheria  bacilli  are  fairly 
numerous  and  appear  to  be  responsible,  in  part  at  least,  for  the 
spread  of  the  disease.  From  1  to  3  per  cent.1  of  healthy  people  harbor 
fully  virulent  bacilli  in  their  mouths,  and  about  2  per  cent,  of  all 
school  children  in  large  cities  have  them.  Positive  results  may  also 
be  obtained  with  avirulent  strains  of  diphtheria  bacilli.  In  order  to 
determine  the  virulence  it  is  necessary  to  isolate  the  organism  in 
pure  culture  and  to  inject  two  guinea-pigs  respectively  with  a  forty- 
eight-hour  broth  culture.  The  isolation  is  best  made  from  cultures 
on  Loffler's  blood  serum  which  microscopic  examination  has  shown 
to  contain  diphtheria  bacilli.  Such  a  culture  is  emulsified  in  broth 
and  streaked  out  on  an  agar  plate,  or,  better,  upon  blood  agar  plates. 
After  twenty-four  hours'  incubation  diphtheria  colonies  are  removed 
to  plain  (sugar-free)  broth  and  incubated  two  days.  One-half  a  cubic 
centimeter  of  this  forty-eight-hour  broth  culture  per  100  grams  weight 
of  guinea-pig  is  injected  into  Pig  A,  and  a  similar  amount  of  the  broth 
culture,  mixed  prior  to  inoculation  with  an  excess  of  antitoxin,  allow- 
ing half  an  hour  for  the  antitoxin  to  unite  with  the  toxin  prior  to 
inoculation,  injected  into  guinea-pig  B.  Guinea-pig  A  should  die 
in  from  one  to  five  days,  and  an  autopsy  should  present  a  typical 

1  Recent  observations  by  Moss,  Guthrie,  and  Gelien  (Tr.  XV  Congress  on  Hyg. 
and  Demog.,  1912,  iv,  156)  indicate  that  the  number  of  carriers  of  virulent  diphtheria 
bacilli  may  greatly  outnumber  the  actual  cases  of  the  disease.  Their  observations 
showed  that  carriers  were  about  four  times  as  numerous  as  the  cases. 


THE  DIPHTHERIA   BACILLUS  403 

picture  of  diphtheria  poisoning.  Guinea-pig  B  should  live  because 
the  diphtheria  toxin  is  neutralized  by  the  antitoxin. 

The  diagnosis  of  diphtheria  by  serological  methods  is  not  practical. 

Dissemination  and  Prophylaxis. — Diphtheria  bacilli  are  spread  chiefly 
by  contact  or  by  carriers.  Occasionally  milk  appears  to  be  a  vehicle 
of  transmission.  As  a  prophylactic  agent  for  destroying  diphtheria 
bacilli,  antitoxin  is  one  of  the  greatest  blessings  which  bacteriology  has 
conferred  on  medicine.  Diphtheria  antitoxin  is  used  in  two  ways :  (a) 
prophylactically ;  (6)  curatively.  If  diphtheria  breaks  out  in  a  house- 
hold or  a  hospital,  those  in  contact  with  the  patient  should  receive  pro- 
phylactic doses  of  antitoxin:  that  is,  from  500  to  1500  units  of  antitoxin 
repeated  after  fourteen  days  or  until  all  danger  is  over,  provided 
the  Schick  test  is  faintly  or  markedly  positive.  (See  Schick  test.) 
Curatively,  from  3000  to  15,000  units,  or  in  severe  cases  20,000  units, 
or  even  more,  are  used.  In  severe  and  desperate  cases  the  antitoxin 
should  be  introduced  intravenously,  preferably  using  antitoxin  pre- 
pared without  preservatives  for  this  purpose.  Antitoxin  must  be 
used  early.  If  it  is  used  early  the  mortality  is  reduced  more  than  50 
per  cent.  If  the  serum  is  used  within  the  first  twenty-four  hours, 
the  prognosis  is  favorable  in  at  least  95  per  cent,  of  the  cases.  The 
general  death  rate  prior  to  the  introduction  of  antitoxin  was  from 
25  to  33  per  cent.;  since  the  use  of  antitoxin  it  varies  from  3  to  14  per 
cent. 

In  a  certain  proportion  of  cases  of  diphtheria  treated  with  anti- 
toxin, usually  from  eight  to  fourteen  days  after  the  administration 
of  antitoxin,  rashes  and  painful  joints  develop,  together  with  fever, 
angioneurotic  edema,  swollen  lymph  glands,  and  albuminuria.  This 
is  the  so-called  serum  disease,  which  is  usually  particularly  severe  in 
asthmatics,  in  whom  there  occasionally  develops  a  true  bronchial 
spasm  with  respiratory  embarrassment.  In  a  few  cases,  less  than  one 
in  ten  thousand,  sudden  death  may  occur  within  five  to  fifteen  minutes 
after  the  injection.  At  autopsy  there  is  usually  found  a  persistent 
thymus.  These  are  cases  of  status  lymphaticus.  This  sudden  death 
is  not  due  to  the  antitoxin,  but  to  the  proteins  in  the  horse  serum  in 
which  the  antitoxin  is  contained.1  If  there  is  reason  to  suspect  that 
the  administration  of  antitoxin  will  result  seriously,  a  few  drops  (not 
more  than  a  quarter  of  a  cubic  centimeter)  should  be  injected,  and 

1  It  should  be  remembered  in  this  connection  that  man  is  less  susceptible  than  a 
guinea-pig  to  serum  diseases,  and,  furthermore,  it  ordinarily  takes  about  5  c.c.  of  horse 
serum  to  bring  about  the  anaphylactic  reaction  in  sensitized  guinea-pigs.  Proportion- 
ately, it  would  take  200  c.c.  to  induce  the  same  symptoms  in  man. 


404  THE  DIPHTHERIA   BACILLUS  GROUP 

the  remainder  after  half  an  hour.  The  first  small  injection  indicates 
the  susceptibility  of  the  patient;  if  no  symptoms  appear  the  full  dose 
may  be  given  with  impunity;  even  if  symptoms  do  appear  the  anaphy- 
lactic  shock  is  aborted  by  the  first  injection  and  the  remainder  may 
be  given  at  the  end  of  an  hour. 

BACILLI   SIMILAR   TO   THE   DIPHTHERIA   BACILLUS. 

There  is  a  group  of  bacteria  closely  related  to  Bacillus  diphtherise, 
but  differing  from  it  either  in  virulence,  morphology,  or  both.  Certain 
of  these  organisms  exhibit  the  characteristic  morphology,  staining  and 
cultural  reactions  of  the  diphtheria  bacillus,  but  do  not  form  toxin; 
these  strains,  which  are  occasionally  found  in  healthy  and  diseased 
throats,  may  be  tentatively  regarded  as  non-toxin-producing  variants 
of  the  type  organism. 

In  addition  to  the  non-virulent  but  morphologically  typical  diph- 
theria bacilli,  other  bacteria  have  been  described  which  resemble 
Bacillus  diphtheriae  superficially,  but  differ  from  it  in  certain  impor- 
tant details.  Two  principal  types  have  been  recognized:  Bacillus 
hofmanni  and  Bacillus  xerosis. 

Bacillus  Hofmanni. — Bacillus  hofmanni  appears  to  have  been 
first  observed  by  Loffler;1  somewhat  later  Hofmann2  studied  it  in 
considerable  detail. 

Morphologically  the  Hofmann  bacillus  is  somewhat  shorter  and 
relatively  thicker  than  Bacillus  diphtherise,  and  more  uniform  in  size 
and  shape.  Stained  with  Loffler 's  methylene  blue,  but  a  single 
unstained  area  is  observed  typically,  the  organism  being  somewhat 
diplococcoid  in  form  under  these  conditions. 

Growth  is  relatively  more  luxuriant  in  artificial  media  than  that 
of  the  diphtheria  bacillus,  and  no  toxin  is  produced  in  sugar-free  broth. 
The  organism  ferments  no  sugars,  not  even  dextrose. 

Bacillus  hofmanni  is  found  not  infrequently  in  normal  and  diseased 
throats,  and  occasionally  in  the  nasal  secretion. 

Bacillus  Xerosis. — Bacillus  xerosis,  first  observed  by  Bezold,3  was 
obtained  in  pure  culture  from  several  cases  of  a  chronic  type  of  con- 
junctivitis known  as  xerosis  by  Kirschbert  and  Neisser.4  Recently 
the  organism  has  been  isolated  repeatedly  from  the  healthy  conjunc- 

1  Centralbl.  f.  Bakt.,  1887,  ii,  106. 

2  Wien.  klin.  Wchnschr.,  1888,  Nos.  3  and  4. 
sBerl.  klin.  Wchnschr.,  1874,  p.  408. 

4  Breslauer  arztl.  Ztschr.,  1883,  No.  4. 


BACILLI  SIMILAR  TO  THE  DIPHTHERIA  BACILLUS       405 

tiva  and  the  nasal  secretion.  The  morphological  similarity  between 
Bacillus  hofmanni  and  Bacillus  xerosis  has  doubtless  led  to  confusion 
in  the  past.  Knapp1  has  studied  the  fermentation  reactions  of  the 
group  and  has  shown  that  within  the  diphtheria  group  three  cultural 
types  are  recognizable,  as  follows: 

Dextrose.  Saccharose. 

Bacillus  diphtheria?   ..    .    U      .     1     .     V     .    acid"  alkaline 

Bacillus  hofmanni      .  '    .      .      ...      .      .    alkaline  alkaline 

Bacillus  xerosis    .-     .      .      .      .            .      -     :-    acid  acid 

Bacillus  Hodgkini.— Hodgkin's  disease,  a  malignant  granulomatous 
lymphatic  infection  long  regarded  as  a  special  type  of  infection  with 
the  tubercle  bacillus,  is  now  generally  regarded  as  an  infectious  entity 


FIG.  57.— Pseudodiphtheria  bacilli.     (Park.) 

quite  apart  from  tuberculosis.  The  etiology  remained  obscure  until 
Negri  and  Mieremet2  published  a  description  of  a  pleiomorphic,  diph- 
theroid  bacillus  obtained  from  two  undoubted  cases.  The  organism, 
which  was  found  to  be  Gram-positive,  received  the  name  Corynebac- 
terium  granulomatis  maligni.  Bunting  and  Yates3  have  recovered 
a  similar  pleiomorphic  bacillus  from  several  cases  of  Hodgkin's  disease. 
Initial  cultures  were  obtained  upon  Dorset's  egg  medium.  Subse- 
quent development  upon  ordinary  media  gave  the  following  cultural 
reactions:  gelatin  not  liquefied;  little  or  no  change  in  litmus  milk; 
an  adherent  growth  in  broth  tubes  with  the  gradual  accumulation  of 
a  slimy  sediment.  The  colonies  upon  serum  and  agar  are  not 
characteristic. 

1  Jour.  Med.  Research,  November,  1904,  vol.  xii,  475. 

2  Centralbl.  f.  Bakt.,  Orig.,  1913,  Ixviii,  292. 

3  Arch.  Int.  Med.,  1913,  xii,  236.     See  also  Bull.  Johns  Hopkins  Hosp.,  1915,  xxvi, 
376,  for  relation  of  pseudodiphtheria  bacilli  to  leukemia,  pseudoleukemia,  and  Banti's 
disease. 


406  THE  DIPHTHERIA   BACILLUS  GROUP 

Morphologically  the  organism  is  variable  in  shape.  Bacillary 
forms  predominate  in  young  cultures,  but  the  bacilli  exhibit  a  marked 
tendency  toward  coccoid  elements  after  prolonged  cultivation. 

The  etiological  relationship  of  the  organism,1  which  received  the 
name  Corynebacterium  hodgkini,  to  Hodgkin's  disease  is  as  yet  un- 
determined, but  vaccines  injected  into  several  typical  cases  caused 
a  definite  recession  in  the  size  of  the  enlarged  glands.  The  permanence 
of  this  recession  must  await  final  reports. 

Bunting  and  Yates2  injected  their  organism  into  monkeys,  and  a 
chronic  lymphadenitis  with  an  increased  mononu clear  and  eosinophile 
count  resulted.  A  polymorphonuclear  leukocytosis  was  not  observed. 
They  conclude  that  the  anatomical  lesions  were  very  similar  to  those 
observed  in  the  early  stages  of  Hodgkin's  disease  in  man. 

1  See  excellent  resume  by  Bloomfield,  Arch.  Int.  Med.,  August,  1915,  p.  197. 

2  Jour.  Am.  Med.  Assn.,  1913,  Ixi,  1803;    ibid.,  1914,  Ixii,  516. 


CHAPTER  XXI. 

THE  HEMORRHAGIC  SEPTICEMIA  GROUP. 

THE  Hemorrhagic  Septicemia  or  Pasteurella  Goup  of  bacilli  com- 
prises a  number  of  organisms  which  possess  in  common  peculiarities 
of  morphology,  similarity  of  cultural  characters  and  great  patho- 
genicity  for  animals. 

Morphologically  they  are  short,  ovoid  bacilli  of  relatively  large 
diameter,  measuring  about  0.5  to  0.8  micron  in  the  widest  part,  and 
varying  in  length  from  0.8  to  1.5  microns.  The  organisms  usually 
exhibit  marked  pleiomorphism  in  old  lesions  and  in  old  cultures. 
They  are  non-motile,  uniformly  Gram-negative,  and  exhibit  a  marked 
tendency  to  bipolar  staining;  the  stainable  substance  is  collected  at 
the  ends  of  the  bacillus,  separated  by  a  central,  faintly  stainable  area. 

The  hemorrhagic  septicemia  bacilli  grow  well  upon  ordinary  cul- 
tural media,  and  they  are  chemically  relatively  inert.  Indol  is  pro- 
duced by  certain  types,  but  not  by  all.  Gelatin  is  not  liquefied.  Acid, 
but  no  gas,  is  formed  in  dextrose,  lactose  and  many  hexoses.  The 
fermentation  of  other  sugars  and  starches  has  not  been  thoroughly 
studied. 

The  type  of  infection  induced  is  usually  an  acute  generalized 
septicemia,  which,  because  of  punctate  hemorrhages  on  serous  sur- 
faces, and  in  the  internal  organs,  is  called  hemorrhagic  septicemia. 
Inflammation  of  the  intestinal  tract  and  frequently  the  respiratory 
tract  is  usually  an  important  feature  of  the  infection. 

The  most  important  animal  diseases  are  chicken  cholera,  swine 
plague,  rabbit  septicemia,  and  a  similar  disease  of  cattle  and  wild 
herbivora.  Plague  is  the  disease  of  man  which  most  closely  approaches 
hemorrhagic  septicemia  of  the  lower  animals. 

The  lesions  caused  by  Bacillus  pestis  in  experimental  animals  and 
in  the  naturally  occurring  disease  in  rodents  present  many  similarities 
to  the  hemorrhagic  septicemias,  and  occasionally  a  distinction  must 
be  made  between  the  plague  bacillus  and  other  members  of  the  group. 
The  Indian  Plague  Commission  state  that  Bacillus  pestis  may  be 
differentiated  from  the  other  members  of  the  group  by  its  ability  to 
develop  and  produce  acid  (but  no  gas)  in  dextrose  and  mannite  media 


408  THE  HEMORRHAGIC  SEPT  ICE  MI  A   GROUP 

containing   bile    salts,    particularly   sodium    taurocholate ;  the   other 
organisms  will  not  grow  in  this  medium. 

Bacillus  Pestis. — Plague,  the  most  dreaded  of  the  acute  epidemic 
diseases,  has  at  somewhat  irregular  intervals  swept  over  parts  of  the 
Orient,  and  during  the  earlier  centuries  of  the  Christian  era  even 
invaded  Europe.  The  great  epidemics  of  the  third  and  the  fourteenth 
centuries  caused  widespread  death;  literally  millions  perished,  and 
the  effect  upon  the  residual  population  was  most  distressing.  The 
disease  has  recently  become  endemic  on  the  western  coast  of  the 
United  States,  the  reservoir  of  infection  being  certain  rodents. 


FIG.  58. — Plague  bacillus,  bouillon  culture,  methyl ene-blue  stain  showing  bipolar 
staining.      X  1000.     (Kolle  and  Hetsch.) 


The  causative  organism,  Bacillus  pestis,  was  isolated  and  described 
almost  simultaneously  by  Kitasato1  and  Yersin2  from  the  purulent 
contents  of  buboes,  the  lymph  glands,  the  blood  and  the  cerebrospinal 
fluid.  Later  the  specificity  of  the  organism  was  established  by  labora- 
tory accidents  and  by  the  very  comprehensive  studies  of  the  British 
Indian  Plague  Commission. 

Morphology. — Bacillus  pestis  is  a  small  thick  bacillus  with  rounded 
ends,  wilich  occurs  singly  or  in  pairs  as  a  rule,  although  short  chains 
of  three  to  six  elements  are  occasionally  seen.  The  organism  is  not 
characteristically  rod-shaped,  rather  it  approaches  in  outline  a  some- 
what ovoid  cell.  The  size  varies  within  the  comparatively  narrow 
limits  of  0.5  to  0.7  micron  in  diameter  at  the  widest  part  and  1.5 
to  1.8  microns  in  length.  The  bacilli  are  very  pleiomorphic  and  exhibit 
great  variation  in  size  and  shape  according  to  the  medium  and  age 

1  Lancet,  1894.  2  Ann.  Inst.  Past.,  1894,  p.  662. 


BACILLUS  PEST  IS  409 

of  the  culture.  In  young  cultures  and  fresh  lesions  the  typical  ovoid 
shape  predominates,  but  in  older  cultures  and  lesions  considerable 
variation  in  size  and  outline  is  very  common.  The  addition  of  2  to 
3  per  cent,  of  salt  to  artificial  media  greatly  increases  the  proportion 
of  involution  forms.  Bacillus  pest  is  is  non-motile  and  possesses  no 
flagella.  Spores  are  not  produced.  Zettnow1  and  Albrecht  and  Ghon2 
state  that  the  organism  forms  a  capsule.  The  organism  stains  readily 
with  anilin  dyes,  and  it  is  Gram-negative.  Dilute  methylene  blue 
colors  the  bacilli  in  a  characteristic  manner.  This  is  best  observed 
when  the  bacilli  are  fixed  with  absolute  alcohol  for  thirty  minutes  in 
place  of  heating.3  The  centre  of  the  cell  is  practically  uncolored  and 
the  stain  able  substance  is  seen  as  a  deeply  colored  granule  at  each 


FIG.  59. — Plague  bacillus.     Involution  forms  from  culture  on  3  per  cent,  salt  agar. 
X  1000.     (Kolle  and  Hetsch.) 

end  of  the  rod — bipolar  staining.     Pleiomorphic  forms  are  usually 
stained  faintly  or  scarcely  at  all  by  this  method. 

Isolation  and  Culture. — The  plague  bacillus  grows  readily  on  ordinary 
media  and  pure  cultures  are  usually  readily  obtained  from  the 
aspirated  contents  of  unopened  buboes  or  other  lesions,  and  frequently 
from  the  blood  stream  in  septicemic  cases.  Colonies  on  agar  after 
twenty-four  hours'  incubation  are  small,  somewhat  irregular  in  out- 
line, translucent,  and  not  distinctive.  Similar  growths  appear  upon 
gelatin  after  two  to  three  days'  incubation.  The  medium  is  not 
liquefied.  The  bacilli  develop  with  considerable  luxuriance  in  broth 
forming  a  granular  sediment  in  the  bottom  of  the  tube  and  frequently 
adhering  to  the  sides.  The  addition  of  a  drop  of  neutral  oil — as  cocoa - 

1  Ztschr.  f.  Hyg.,  1896,  xxi,  165.  2  Centralbl.  f.  Bakt.,  1899,  xxvi,  362. 

3  Kossel  and  Overbeck,  Arb.  a.  d.  kais.  Gesamte,  1901,  xviii,  117. 


410  THE  HEMORRHAGIC  SEPTICEMIA   GROUP 

nut  oil — provided  the  culture  is  maintained  free  from  all  vibration, 
causes  a  characteristic  "stalactite"  growth;  the  organisms  grow 
down  from  the  oil  droplets  as  filaments  (which  have  been  likened  to 
stalactites)  until  they  even  reach  the  bottom  of  the  tube.  Chains  of 
bacilli  are  most  characteristically  developed  in  this  medium.  The 
addition  of  2  to  3  per  cent,  common  salt  to  broth  or  agar  stimulates 
the  formation  of  very  irregular  involution  forms.  Milk  is  not  coagu- 
lated, but  a  slight  permanent  acidity  gradually  develops.  Growth  on 
coagulated  blood  serum  or  ascitic  agar,  although  somewhat  more 
luxuriant  than  on  ordinary  laboratory  media,  is  not  characteristic. 

Bacillus  pestis  is  an  aerobic  organism;  it  fails  to  develop  with  its 
customary  vigor  in  the  absence  of  oxygen.  Unlike  a  majority  of 
pathogenic  bacteria,  the  optimum  temperature  of  growth  is  about  30° 
C.;  growth  ceases  below  10°  C.  and  above  40°  C.  The  viability  of  the 
organism  in  cadavers  is  considerable;  they  may  remain  alive  for 
several  weeks.  In  pus  and  sputum  viable  cultures  may  be  obtained 
after  one  or  even  two  weeks.  Exposure  to  sunlight  kills  the  bacilli 
within  a  few  hours,  and  naked  germs  (unprotected  by  mucus  or 
other  protein  envelope)  are  rapidly  killed  by  drying.  An  exposure 
to  58°  C.  for  an  hour,  or  100°  C.  for  a  few  minutes  is  fatal:  5  per  cent, 
carbolic  acid  and  1  to  1000  bichloride  of  mercury  kill  the  organisms 
within  fifteen  minutes.  The  virulence  of  the  bacilli  diminishes  rather 
rapidly  in  artificial  media  as  a  rule. 

Products  of  Growth. — Indol  is  not  produced  in  sugar-free  broth. 
Acids,  but  no  gas,  are  produced  in  dextrose,  lactose,  galactose,  mannite 
and  maltose,  but  not  in  saccharose,  sorbite,  dulcite,  and  inulin. 

No  enzymes  have  been  demonstrated  in  cultures  of  plague  bacilli. 

Toxins. — Filtered  cultures  of  plague  bacilli  possess  little  or  no 
toxicity,  although  old  broth  cultures,  freed  from  bacteria  by  filtration 
through  unglazed  porcelain,  may  exhibit  slight  toxic  action.  It  is 
probable  that  this  toxicity  is  referable  to  some  endotoxin  which  has 
been  liberated  in  the  medium  during  the  gradual  autolysis  of  the 
organisms.  The  symptoms  of  plague  are  attributed  to  the  action  of 
endotoxins  which  are  liberated  within  the  host  as  the  organisms  dis- 
integrate. The  virulence  of  plague  cultures  is  variable.  Freshly 
isolated  strains  may  occasionally  exhibit  almost  no  virulence  for 
experimental  animals,  although  as  a  rule  they  are  very  virulent. 
Prolonged  cultivation  upon  artificial  media  usually  results  in  a  decided 
lowering  of  virulence,  although  here  again  exceptions  are  met  with.1 

1  McCoy  and  Chapin,  Pub.  Health  Bull..  January,  1912,  No.  53,  p.  1. 


BACILLUS  PESTIS  411 

Pathogenesis. — Animal. — "Plague  is  primarily  a  disease  of  rodents, 
and  secondarily  and  accidentally1  a  disease  of  man."2  The  reservoir 
of  plague  appears  to  be  certain  rodents;  the  disease  exists  in  chronic 
form  in  the  marmot  (Arctomys  bobac)  of  India,  and  has  recently  been 
discovered  in  the  western  United  States  as  a  chronic  disease  in  the 
ground  squirrel  (Citellus  beechyi)  by  Wherry,3  whose  observations 
have  been  confirmed  by  McCoy.  McCoy4  and  Chapin5  have  found 
that  a  smaller  member  of  the  squirrel  family,  Ammospermophilus 
lecurus)  is  also  susceptible  to  infection  with  Bacillus  pestis.  The 
various  members  of  the  genus  Mus — Mus  norwegicus,  Mus  rattus, 
Mus  alexandrinus,  and  probably  Mus  musculus,  "are  the  producers 
of  acute  outbreaks,  the  conduit  for  the  carriage  of  the  virus  from  its 
perpetuating  reservoir  to  the  body  of  man."  Guinea-pigs  are  some- 
what more  susceptible  to  infection  with  the  plague  bacillus  than 
rodents;  the  disease  appears  to  have  a  seasonal  distribution  among 
rodents6  in  India,  and  these  epidemic  periods  coincide  in  time  with 
epidemics  in  man.  Immediately  following  epidemic  periods  con- 
siderable numbers  of  rodents  appear  to  be  relatively  non-susceptible 
to  infection.  Rabbits7  and  monkeys8  are  also  susceptible.  Dogs 
and  cats  are  more  refractory;  herbivora  appear  to  be  practically 
immune,  at  least  to  natural  infection. 

The  lesions  observed  in  rats  are  striking  and  important  because 
plague  epidemics  usually  appear  about  two  weeks  earlier  among 
these  animals  than  in  man.  Infection  may  take  place  through  infected 
fleas  from  other  rats,  from  ingestion  of  dead  plague-infected  animals, 
or  by  inhalation.  The  lesions  are  those  of  an  hemorrhagic  septicemia; 
upon  laying  open  the  animal,9  the  inguinal  and  axillary  glands  are 
usually  enlarged  (buboes),  markedly  injected  and  frequently  hemor- 
rhagic. The  contents  may  be  firm,  or,  less  commonly,  purulent. 
The  peritoneal  and  pleufal  surfaces  are  red  and  injected,  and  there 
is  an  excess  of  fluid  in  both  cavities.  The  spleen  is  enlarged,  engorged 
and  moderately  soft,  and  the  liver  usually  presents  a  mottled  appear- 
ance, due  to  punctiform  hemorrhages  and  areas  of  necrosis  which 

1  This  statement  may  possibly  require  modification  in  connection  with  the  pneumonic 
type  of  plague  in  man  (see  human  pathogenesis) . 

2  Rucker,  Public  Health  Reports,  July  19,  1912,  p.  1130. 

3  Public  Health  Reports,  1908,  xxiii,  1289;   Jour.  Inf.  Dis.,  1908,  v,  485. 

4  Public  Health  Bull.,  April,  1911,  No.  43. 

5  Ibid.,  January,  1912,  No.  53,  p.  15. 

6  See  Jour.  Hyg.,  1908,  viii,  266,  for  details. 

7  McCoy,  Public  Health  Bull.,  April,  1911,  No.  43. 

8  Wyssokowitsch  and  Zabolotny,  Ann.  Inst.  Past.,  1897,  xi,  665. 

9  Which  should  be  done  after  treatment  with  an  insecticide  to  kill  ecto-parasites. 


412  THE  HEMORRHAGIC  SEPTICEMIA   GROUP 

appear  yellowish  in  contrast  to  the  hemorrhagic  points.  A  simple 
inspection  suffices  as  a  rule  to  establish  a  correct  diagnosis,  although 
cultures  and  smears  should  be  prepared.  Occasionally  rats  are  sub- 
mitted for  diagnosis  which  are  badly  decomposed.  The  rapid  over- 
growth of  adventitious  bacteria  makes  the  isolation  of  Bacillus  pestis 
difficult  by  ordinary  methods.  Albrecht  and  Ghon1  have  shown  that 
the  plague  bacillus,  even  in  the  presence  of  large  numbers  of  con- 
taminating bacteria,  may  be  obtained  in  pure  culture  by  rubbing  the 
suspected  material  upon  the  freshly  shaved  abdomen  of  a  guinea- 
pig.  The  plague  bacillus  readily  penetrates  the  skin  and  causes  a 
rapidly  fatal  generalized  infection  with  characteristic  lesions.  It  may 
be  obtained  in  pure  culture  from  the  internal  organs.  Fritsche2  has 
found  that  other  bacteria,  even  of  the  hemorrhagic  septicemia  group, 
fail  to  penetrate  the  skin  and  infect  the  animal.  This  cutaneous  test 
is  of  great  diagnostic  importance. 

McCoy  and  Chapin3  have  described  a  disease  superficially  resem- 
bling plague  in  its  pathological  anatomy  caused  by  Bacillus  tularense. 
The  disease  is  readily  transmitted  to  guinea-pigs,  rabbits  and  mice, 
less  readily  to  rats.  Wherry  and  Lamb4  have  recently  isolated  the 
organism  from  an  epizootic  among  wild  rabbits  and  from  a  human 
case  presenting  corneal  ulcerations  and  lymphadenitis. 

Man. — The  atria  of  infection  are  chiefly  the  skin  and  the  respira- 
tory tract,  giving  rise  to  two  general  types  of  the  disease,  glandular 
and  pneumonic  plague.  Rarely  a  localized  cutaneous  lesion,  plague 
carbuncle,  is  met  with  where  the  focus  of  localization  of  the  organisms 
appears  to  be  very  circumscribed.  Cases  of  pneumonic  plague  which 
develop  sporadically  during  epidemics  of  the  bubonic  type  do  not  as 
a  rule  appear  to  spread  rapidly;  on  the  contrary,  during  epidemics 
in  which  the  pneumonic  type  predominates  the  infectivity  from  man 
to  man  is  very  great.  No  theory  has  been  presented  in  explanation 
of  this  very  unusual  phenomenon,  and  the  origin  of  the  pneumonic  type 
of  the  disease  is  not  definitely  known.5  Typical  pneumonic  plague 
resembles  lobar  pneumonia  in  its  symptomatology,  and  the  fatalities 

1  Denkschrift    der   math-Naturw.   Klasse  der  kaiserl.   Akad.   d.   Wissensch.,   Wicn., 
1898,  Ixvi. 

2  Arb.  a.  d.  kais.  Gesamte,  1902,  vol.  xviii. 

3  Jour.  Inf.  Dis.,  1912,  x,  61;  Public  Health  Bull.,  April,  1911,  No.  43;  ibid.,  January, 
1912,  53. 

4  Jour.  Inf.  Dis.,  1913,  xv,  331 ;   Jour.  Am.  Med.  Assn.,  1914,  Ixiii,  2041. 

6  Animal  experiments  suggest  that  attenuated  cultures  of  Bacillus  pestis  which  fail 
to  kill  guinea-pigs  by  subcutaneous  inoculation  may  give  rise  to  a  fatal  infection  when 
inoculated  into  the  respiratory  tract,  and  that  the  virulence  of  cultures  may  be  recovered 
by  this  process.  The  high  mortality  observed  during  epidemics  of  pneumonic  plague 
in  man  may  be  a  similar  phenomenon. 


BACILLUS  PESTIS  413 

are  very  great.  Death  usually  intervenes  in  less  than  a  week.  The 
marked  cardiac  depression  which  is  a  feature  of  this  type  of  plague 
is  of  some  differential  diagnostic  value.  One  or  more  lobes  are  infected, 
the  inflammation  being  catarrhal  in  nature.  Enormous  numbers  of 
plague  bacilli  are  coughed  up  with  the  sanguinoserous  exudate,  which 
are  readily  transmitted  to  doctors,  nurses  and  attendants  by  droplet 
infection.  Very  frequently  a  generalized  invasion  of  the  blood  stream 
occurs. 

Bubonic  plague,  the  most  common  type  of  the  disease  in  man,  is 
essentially  a  localization  of  plague  bacilli  which  have  gained  entrance 
to  the  tissues  through  the  skin  in  the  regional  lymph  glands.  The 
inguinal  glands  are  more  commonly  invaded;  next  in  order  of  fre- 
quency are  the  axillary,  then  the  cervical  glands.  The  glands 
are  violently  inflamed,  and  not  infrequently  soften  and  caseate.  A 
generalized  blood  infection — septicemic  plague — may  occur  either 
secondarily  following  the  development  of  a  bubo  or  of  pneumonic 
plague,  or,  less  commonly,  as  an  initial  generalized  invasion  following 
very  shortly  after  infection  and  before  the  bubo  becomes  conspicuous. 
In  such  cases  the  organisms  may  be  obtained  in  pure  culture  from  the 
blood  stream,  and  in  about  20  per  cent,  of  cases  may  be  actually 
demonstrated  in  stained  preparations  of  the  blood  by  microscopical 
examination. 

Immunity  and  Immunization. — Recovery  from  one  attack  of  plague 
in  man  almost  always  confers  lasting  immunity.  Immunity  has  been 
induced  in  animals — monkeys,  rats  and  guinea-pigs — by  inoculation 
with  living  avirulent  cultures.  Usually  a  moderate  reaction  is  noticed, 
and  a  bubo  may  even  form,  but  the  animal  recovers,  and  even  after 
months  successfully  resists  several  times  the  fatal  dose  of  virulent 
organisms.  This  method  is  far  too  dangerous  for  human  practice. 
Bacillus  pseudo tuberculosis  rodentium,  an  organism  that  occasionally 
is  found  in  diseased  rats  producing  lesions  superficially  not  unlike 
plague,  will  immunize  rats  against  Bacillus  pestis  and  vice  versa.  This 
bacillus  must  be  sharply  differentiated  from  the  plague  bacillus  in 
the  microscopic  diagnosis  of  plague  in  rodents.  It  fails  to  infect  guinea- 
pigs  by  the  cutaneous  method,  however,  and  is  less  virulent  for  labora- 
tory animals.  Cultures  of  plague  bacilli  heated  to  50°  C.,  or  killed 
with  chemicals,  as  phenol  or  alcohol,  have  also  been  employed  success- 
fully, but  the  degree  of  resistance  to  subsequent  infection  is  less.1 
Specific  bacteriolysins  and  agglutinins  develop  during  the  immunizing 
process.  . 

1  Kolle  and  Otto,  Ztschr.  f.  Hyg.,  1903,  xlv,  507. 


414  THE  HEMORRHAGIC  SEPTICEMIA   GROUP 

Active  immunization  of  man  against  plague  has  been  accomplished 
by  Haffkine,  using  broth  cultures  of  plague  bacilli  grown  in  shallow 
layers  of  broth  containing  droplets  of  cocoanut  or  other  neutral  oil 
on  the  surface  to  increase  the  development  of  the  organisms  by  stalac- 
tite formation;  after  about  six  weeks'  incubation,  during  which  time 
several  crops  of  bacilli  develop  and  sink  to  the  bottom,  the  culture 
is  heated  to  60°  to  65°  C.  for  an  hour,  and  0.5  per  cent,  phenol  is 
added.  2  to  3.5  c.c.  of  the  killed  culture  are  injected  subcutaneously 
into  adults,  proportionately  smaller  amounts  into  children.  Usually 
a  second  injection  is  given,  somewhat  larger  in  amount,  after  ten 
days.  The  German  Plague  Commission1  used  forty-eight-hour  agar 
cultures  of  virulent  plague  bacilli  emulsified  in  salt  solution  and 
sterilized  at  65°  C.;  0.5  per  cent,  phenol  was  added  as  a  preservative. 
The  amount  for  injection  into  an  adult  was  the  equivalent  of  one 
agar  culture  of  the  organism.  Available  evidence  indicates  that 
prophylactic  inoculation  against  plague  reduces  materially  both  the 
morbidity  and  mortality  of  the  disease.  The  protection,  as  the 
statistics  show,  is  by  no  means  absolute,  and '  it  appears  that  the 
duration  of  resistance  to  infection  is  indeterminate,  probably  on  the 
average  several  months.  A  serum  obtained  from  horses  immunized 
against  plague  bacilli  has  been  prepared,  but  its  use  in  man  has  on 
the  whole  been  irregular  and  disappointing;  the  chief  practical  use 
appears  to  be  in  those  cases  where  exposure  to  infection  is  reason- 
ably certain,  as  for  example,  in  those  attending  plague  patients.  The 
excessive  cost  of  the  serum  is  prohibitive  for  general  use. 

Transmission  and  Plague. — The  Interim  Report  of  the  Advisory 
Committee  for  Plague  Prevention  in  India2  contains  a  very  excellent 
summary  of  the  mechanism  of  plague  transmission  by  the  flea.  In 
bubonic  plague,  the  most  common  type  seen  in  man,  the  plague  bacilli 
are  locked  up  in  the  body,  as  it  were,  and  can  not  of  themselves  escape 
to  other  hosts.  The  rat  is  usually  the  source  of  infection  in  plague 
epidemics,  and  rat  fleas,  Xenopsyllus  cheopis,  transmit  the  disease 
from  rat  to  rat  and  from  rat  to  man.  When  the  host  dies  (rat  or  man) 
its  ectoparasites  escape  if  possible  to  living  hosts.  It  was  shown  by 
the  Indian  Commission3  that  fleas  from  plague-infected  rats  frequently 
contained  large  numbers  of  plague  bacilli  in  their  intestinal  tracts, 

1  Gaffky,  Pfeiffer,  Sticker,  and  Dieudonne,  Bericht  u.  d.  Thatigkeit  dcr  zur  Erfor- 
schung  der  Pest  im  Jahre  1897,  etc.,  Berlin,  1899. 

2  Jour.  Hyg.,  1910,  x,  No.  3. 

3  See  Jour.  Hyg.,  1906,  1907,  1908,  1910,  for  a  most  complete  discussion  of  the  relation 
of  fleas  to  the  transmission  of  plague. 


BACILLUS  PESTIS  415 

and  that  the  bacilli  were  present  at  least  three  weeks  after  the  last 
feeding.  In  the  absence  of  fleas  no  infection  takes  place,  at  least  in 
man.  The  bite  of  the  infected  flea  may  result  in  infection,  or,  since 
the  feces  of  the  flea  are  usually  deposited  during  feeding,  laden  with 
plague  bacilli,  the  irritant  flea  bite  may  lead  to  scratching  of  the  area, 
resulting  in  the  "rubbing  in"  of  the  bacilli  deposited  with  the  rat 
feces.  Epidemics  of  pneumonic  plague  are  spread  by  droplet  infection. 
Preventive  measures  include  the  appropriate  care  of  the  patient  and 
measures  to  reduce  the  rat  population.  This  is  accomplished  by  careful 
disposal  of  all  garbage,  rat  proofing  all  houses  and  granaries,  and  an 
active  campaign  against  rats  by  poison,  destruction  of  nests  and  run- 
ways, and  the  creation  of  rodent-free  zones  of  considerable  magnitude 
around  settlements. 


FIG.  60. — Influenza  bacillus  from  sputum.      X  1200.     (Kolle  and  Hetsch.) 

Bacteriological  Diagnosis. —  Human— The  juice  of  buboes,1  of  lymph 
glands,  of  petechiae,  the  blood,  the  sputum  from  pneumonic  cases, 
and  occasionally  the  urine  contain  plague  bacilli  in  large  numbers. 
They  may  be  obtained  readily  from  the  spleen,  liver,  lungs  and  kidneys 
of  the  cadaver. 

Animal. — The  postmortem  appearance  of  plague-infected  rats  is 
very  characteristic. 

Microscopical  Diagnosis. — The  presence  of  Gram-negative  ovoid 
short  bacilli  in  considerable  numbers  in  films  prepared  from  material 
outlined  above  is  very  suggestive,  but  not  conclusive  evidence  of 
infection  with  Bacillus  pestis.  In  man  the  evidence  is  stronger  than 

1  Buboes  which  have  suppurated  frequently  do  not  contain  plague  bacilli,  or  plague 
bacilli  in  association  with  extraneous  organisms.  Even  if  buboes  have  not  developed, 
the  lymphatic  glands  usually  contain  the  bacilli. 


416  THE  HEMORRHAGIC  SEPTICEMIA   GROUP 

in  rats,  for  in  the  latter  Bacillus  pseudotuberculosis  rodentium,  Bacil- 
lus tularense,  and  other  organisms  may  be  present,  which  produce 
lesions  superficially  not  unlike  those  of  plague. 

Cultural  Diagnosis. — Prepare  agar  plates  from  the  contents  of 
enlarged  glands  or  other  material  incubate  at  30°  C.  (or  37°  C.), 
and  isolate  colonies  in  pure  culture.  Blood,  collected  aseptically, 
should  be  plated  out  on  agar.  From  the  pure  colonies  inoculate  3 
per  cent,  salt  agar  and  examine  after  twenty-four  hours  for  involution 
forms;  make  the  "stalactite"  test  in  broth  containing  .a  few  drops  of 
neutral  oil.  (The  culture  must  be  kept  in  an  absolutely  quiet  environ- 
ment to  obtain  stalactite  growth.)  This  reaction  is  not  absolutely 
distinctive  for  other  members  of  the  Hemorrhagic  Septicemia  Group 
may  also  develop  in  this  manner. 

Animal  Inoculation. — A  small  amount  of  culture  inoculated  at  the 
root  of  the  tail  of  a  rat  subcutaneously  or  intranasally  in  a  guinea- 
pig  will  cause  death  within  three  to  five  days  with  characteristic 
lesions.  The  organism  may  be  recovered  from  the  internal  organs. 
If  the  material  for  inoculation  be  mixed  with  adventitious  bacteria, 
the  cutaneous  method  of  inoculation1  of  guinea-pigs  will  give  positive 
results  and  the  organisms  may  be  recovered  in  pure  culture  from  the 
internal  organs  postmortem. 

1  Albrecht  and  Ghon,  loc.  cit. 


CHAPTER  XXII. 

HEMOGLOBINOPHILIC  BACILLI:    KOCH-WEEKS, 
MORAX-AXENFELD  AND  DUCREY  BACILLI. 

BACILLUS   INFLUENZA. 

BACILLUS  INFLUENZA  was  isolated  in  pure  culture  and  described  by 
Pfeiffer.1 

Morphology. — It  is  an  extremely  small  bacillus,  one  of  the  smallest 
known,  measuring  from  0.2  to  0.3  micron  in  diameter  and  from  0.5 
to  1  micron  in  length.  The  ends  are  rounded  and  it  occurs  singly  or 
in  pairs,  rarely  in  short  chains.  The  organism  is  non-motile,  and  no 
flagella  have  been  demonstrated.  Spores  and  capsules  are  not  pro- 
duced. Ordinary  anilin  dyes  do  not  color  the  organism  readily, 
but  Pfeiffer2  has  shown  that  dilute  carbol-fuchsin3  stains  it  readily. 
StaineoTwith  methylene  blue  or  dilute  carbol-fuchsin,  the  ends  of  the 
bacilli  are  colored  somewhat  more  deeply  than  the  centre,  suggesting 
a  bipolar  distribution  of  the  cytoplasm  similar  to  that  exhibited  by 
the  bacteria  of  the  hemorrhagic  septicemia  group.  The  organism  is 
Gram-negative. 

Isolation  and  Culture. — Bacillus  influenzse  is  an  obligately  hemoglo- 
binophilic  organism;  it  does  not  grow  outside  the  body  in  the  absence 
of  hemoglobin,  although  the  amount  of  this  substance  required  to 
encourage  development  may  be  so  small  in  amount  that  it  is  invisible 
to  the  eye.4  The  organism  may  be  isolated  from  bronchial  mucus 
by  Pfeiffer's  method.  The  mucus  is  washed  several  times  with  sterile 
water  to  remove  extraneous  bacteria,  then  spread  upon  blood  agar 
plates.  Human,  pigeon  or  rabbit  blood  added  to  neutral  plain  agar 
creates  a  favorable  medium  for  the  bacillus.  The  colonies  which 
appear  after  twenty-four  to  forty-eight  hours'  incubation  at  37°  C. 
are  very  minute,  clear  and  colorless.  They  may  require  a  lens  for 
their  recognition.  The  hemoglobin  is  not  visibly  changed  in  appear- 
ance and  no  hemolysis  occurs.  Massive  cultures  of  influenza  bacilli 

1  Deutsch.  med.  Wchnschr.,  1892,  No.  2. 

2  Ztschr.  f.  Hyg.,  1893,  xiii,  357. 

3  One  part  carbol-fuchsin,  9  parts  water. 

*Ghon  and  Preyss,  Centralbl.  f.  Bakt.,  1902,  xxxii,  90;    1904,  xxxv,  531. 

27 


418  HEMOGLOBINOPHILIC  BACILLI 

may  be  obtained  in  blood  bouillon.  One  c.c.  of  sterile  defibrinated 
pigeon  or  rabbit  blood  is  added  to  50  c.c.  of  neutral  nutrient  broth. 
After  incubation  to  demonstrate  its  sterility,  the  medium  is  ready 
for  inoculation.1  Attempts  to  grow  the  bacilli  on  hemoglobin-free 
media  have  been  uniformly  negative;  no  development  occurs  in 
ordinary  media. 

Bacillus  influenzse  is  an  aerobic  bacillus.  It  has  not  been  grown  in 
the  absence  of  oxygen.  Growth  does  not  take  place  below  25°  C.  nor 
above  42°  to  43°  C.  The  optimum  temperature  is  37°  C.  It  is  possible 
to  maintain  cultures  by  transplanting  them  upon  fresh  hemoglobin 
media  at  intervals  not  greater  than  five  days.  Drying  is  rapidly  fatal 
to  influenza  bacilli;  dried  in  mucus  the  organisms  are  not  viable 
after  one  to  three  days.  They  may  remain  alive  in  moist  mucus  for 
nearly  two  weeks,  however.  Ten  minutes'  exposure  at  57°  C.  kills 
them  and  ordinary  chemical  disinfectants,  bichloride  of  mercury  1  to 
1000,  and  5  per  cent,  carbolic  acid,  destroy  them  in  a  few  minutes. 

Products  of  Growth. — The  nature  of  the  products  of  metabolism  of 
the  influenza  bacillus  are  unknown.  Enzymes  have  not  been  detected 
in  cultures  of  tjhe  organism  and  soluble  toxins  have  not  been  demon- 
strated. There  is  evidence  that  the  cell  substance  of  the  bacilli  is 
toxic;  it  is  probable  that  this  toxic  substance  is  endotoxic  in  character. 

Pathogenesis. — The  direct  evidence  of  the  etiological  relationship 
of  B.  influenza?  to  the  disease  influenza  rests  upon  a  single  laboratory 
infection  with  a  pure  culture  of  the  organism.  The  hands  were  con- 
taminated, and  within  twenty-four  hours  a  typical  attack  of  influenza 
developed.  The  organisms  persisted  in  the  sputum  for  two  months.2 

Animal. — Influenza  is  essentially  a  disease  of  man.  Pfeiffer3  has 
shown  that  mice,  rats,  guinea-pigs,  swine,  dogs,  and  cats  are  refrac- 
tory to  infection  with  the  living  organisms.  The  introduction  of 
hemoglobin  broth  cultures  of  B.  influenzse  through  the  chest  wall  of 
monkeys  frequently  causes  a  transient  febrile  reaction,  and  a  catarrhal 
bronchitis  which,  however,  is  hot  clinically  comparable  to  influenza 
of  man.  The  animals  recovered  rapidly.  Rabbits  are  susceptible  to 
the  endotoxin  of  the  influenza  bacillus.4  The  injection  of  large  num- 
bers of  living  or  killed  organisms  causes  dyspnea  and  a  paralysis  of  the 
leg  muscles.  Frequently  the  animals  die. 


1  Delius  and  Kolle,  Ztschr.  f.  Hyg.,  1897,  xxiv,  327. 

2  Tedesco,  Centralbl.  f.  Bakt.,  Orig.,  1907,  xliii,  323. 

3  Ztschr.  f.  Hyg.,  1893,  xiii,  357. 

4  Pfeiffer,  loc.  cit.;  Cantani,  Ztschr.  f.  Hyg.,  1896,  xxiii,  265. 


BACILLUS  INFLUENZA  419 

Man. — Influenza  occurs .  pandemically  at  infrequent  intervals: 
during  interpandemic  intervals  the  organism  causes  somewhat  local- 
ized epidemics  of  "grippe,"  and  not  infrequently  appears  to  be  the 
causative  factor  in  "grippe  colds."  The  bacilli  persist  in  the  respira- 
tory tract  as  "opportunists"  and  are  frequently  detected  in  the  lungs 
of  consumptives.  Invasion  takes  place  through  the  respiratory  tract, 
usually  by  droplet  infection,  and  frequently  spreads  by  continuity 
to  the  lungs,  where  a  purulent  broncho-  or  lobular  pneumonia  develops 
in  typical  cases.  Pleurisy  is  a  frequent  complication,  usually  caused 
by  a  secondary  infection  with  pneumococci  or  streptococci.  The  in- 
fluenza bacillus  rarely  causes  pleurisy.  Enormous  numbers  of  bacilli 
are  coughed  up  in  the  sputum.  The  incubation  period  is  brief,  from 
one  to  three  days  as  a  rule.  Influenzal  meningitis,1  pharyngitis  and 
laryngitis2  and  conjunctivitis3  are  not  uncommon.  The  occurrence 
of  influenza  bacilli  in  the  blood  has  been  a  matter  of  controversy. 
Canon,4  Bruschettini5  and  Ghedini6  have  isolated  bacilli  from  the 
blood  of  patients  at  the  height  of  the  disease  which  they  believe  to  be 
B.  influenzse.  Slawyk7  has  reported  a  case  of  generalized  infection 
with  the  influenza  bacillus  which  would  appear  to  confirm  these 
observations.  Other  investigators  have  questioned  the  accuracy  of 
this  work  and  lay  stress  upon  the  incomplete  diagnosis  of  the  organisms 
obtained  from  the  patients.  The  question  can  not  be  regarded  as 
definitely  settled  at  the  present  time. 

Immunity. — Attempts  to  induce  immunity  in  experimental  animals 
have  been  unsuccessful.  Relapses  are  common  in  man,  and  there  is 
no  evidence  of  immunity  as  the  result  of  recovery  from  the  disease. 

Bacteriological  Diagnosis. — 1.  Sputum  raised  from  the  deeper  air 
passages  is  spread  upon  slides,  air  dried,  fixed,  and  stained  with  dilute 
carbol-fuchsin.  Large  numbers  of  minute  organisms  colored  pink 
with  a  tendency  toward  bipolar  staining  are  suggestive  of  the  influenza 
bacillus.  There  is  no  tendency  toward  a  definite  arrangement  of  the 
bacilli.  They  are  frequently  found  in  leukocytes. 

2.  Cultural. — Blood  agar  plates  are  made  by  depositing  a  generous 
drop  of  human,  rabbit  or  pigeon's  blood  in  the  centre  of  an  agar  plate. 

'Pfuhl,  Ztschr.  f.  Hyg.,  1897,  xxvi,  112;  Frankel.  Ztschr.  f.  Hyg.,  1898,  xxvii,  329; 
Jundell,  Jahrb.  f.  Kinderheilk.,  1904,  lix,  777. 

2  Treitel,  Arch.  f.  Laryngol.,  1902,  xiii,  147. 

'Pretori,  Arch.  f.  Augenheilk.,  1907,  Ivii,  97;  Possek,  Wien.  klin.  Wchnschr.,  1909, 
No.  10. 

4  Deutsch.  med.  Wchnschr.,  1892,  No.  3;   Arch.  f.  Anat.  u.  Phys.,  1893,  cxxxi,  401. 

5  Riforma  Med.,  1893,  viii,  783.  •  Centralbl.  f.  Bakt.,  Orig.,  1907,  xliii,  407. 
7  Ztschr.  f.  Hyg.,  1899,  xxxii,  443. 


420  HEMOGLOBINOPHILIC  BACILLI 

Mucus  raised  from  the  deeper  air  passages  is  thoroughly  washed  in 
sterile  water  and  emulsified  in  broth  or  water,  selecting  for  the  pur- 
pose purulent  masses  by  preference,  and  streaked  out  radially  from  the 
drop  of  blood.  After  twenty-four  to  forty-eight  hours'  incubation 
the  plate  is  examined  with  a  lens  for  very  minute,  clear,  homogeneous 
colonies  which  should  be  removed  to  blood  agar  slants.  When  growth 
occurs  transfer  some  of  it  to  plain  agar.  No  further  development 
occurs  unless  some  blood  has  been  removed  with  the  organisms.  The 
failure  of  the  bacteria  to  develop  on  media  free  from  hemoglobin  is 
distinctive. 

3.  Serological. — The  serum  diagnosis  of  influenza  has  been  unsuc- 
cessful. 

Dissemination  and  Prophylaxis. — Influenza  bacilli  are  distributed 
chiefly  by  droplet  infection.  Carriers  are  said  to  be  common.  Prophy- 
laxis is  the  same  as  for  any  respiratory  disease. 

BACILLUS   PERTUSSIS. 

The  etiology  of  pertussis  (whooping-cough)  has  been  a  subject  of 
controversy  for  several  years.  The  problem  is  complicated  by  the 
rather  general  occurrence  of  influenza-like  bacilli  in  the  sputum  and 
bronchial  exudate  from  cases  of  whooping-cough.  A  clean-cut  dif- 
ferentiation between  these  influenzoid  bacilli  and  Bacillus  pertussis 
described  by  Bordet  and  Gengou1  has  been  difficult  and  has  doubtless 
led  to  confusion  in  the  past.  It  is  now  generally  conceded  that  the 
Bordet-Gengou  bacillus  is  worthy  of  serious  consideration  as  the 
etiological  factor  of  whooping-cough. 

Morphology. — B.  pertussis  is  somewhat  larger  than  B.  influenzse, 
measuring  0.3  micron  in  diameter  and  varying  in  length  from  0.5  to 
1.5  microns,  the  average  length  being  about  1  micron.  It  occurs  singly 
and  in  groups,  less  commonly  in  pairs.  The  organism  has  rounded 
ends;  frequently  it  is  almost  ovoid  in  shape.  The  organism  is  non- 
motile  and  possesses  no  flagella.  Neither  capsules  nor  spores  have 
been  demonstrated.  It  stains  poorly  with  ordinary  anilin  dyes  and 
is  Gram-negative.  Carbol  methylene  blue,  carbol  toluidine  blue  and 
dilute  carbol-fuchsin  stain  it  readily.  Methylene  blue  is  also  a  satis- 
factory stain.  The  organisms  stain  irregularly,  particularly  when 
grown  in  artificial  media.  In  young  cultures  and  in  sputum  they 
appear  frequently  with  the  ends  stained  more  deeply  than  the  centre, 
resembling  in  this  respect  the  influenza  bacillus. 

1  Bull.  Acad.  de  Med.  Belgique,  July,  1906;   Ann.  Inst.  Past.,  1906,  xx,  731. 


BACILLUS  PERTUSSIS  421 

Isolation  and  Culture. — Unlike  the  influenza  bacillus,  B.  pertussis 
can  be  made  to  grow  in  media  which  do  not  contain  hemoglobin.  For 
initial  growths  outside  of  the  human  body,  however,  Bordet  and 
Gengou  have  recommended  a  potato-glycerin-blood  agar  medium 
which  is  claimed  to  be  far  more  efficient  than  blood  agar.1  The  Bor- 
det-Gengou  bacillus  is  more  readily  isolated  from  the  bronchial  secre- 
tion during  the  first  paroxysms2  than  later  in  the  disease.  Cultures 
are  obtained  from  bronchial  mucus  which  has  been  washed  several 
times  in  sterile  water,  then  spread  on  the  surface  of  the  potato  medium 
and  incubated  at  37°  C.  After  twenty-four  to  forty-eight  hours' 
incubation  colonies  appear  as  very  minute,  transparent  growths 
which  resemble  dew  drops;  colonies  of  B.  influenzse  frequently  develop 
at  the  same  time,  but  the  colonies  of  the  latter  are  somewhat  larger 
than  those  of  B.  pertussis.  Secondary  transplantations  of  B.  pertussis 
upon  fresh  potato-glycerin-blood  agar  grow  more  luxuriantly  than 
of  B.  influenzse,  however,  and  after  repeated  transfers  the  Bordet- 
Gengou  bacillus  will  grow  upon  ascitic  agar.  The  influenza  bacillus 
will  not  grow  in  media  free  from  hemoglobin.  Ordinary  media,  unless 
ascitic  fluid  or  blood  serum  is  added,  are  wholly  unsuited  for  the 
growth  of  B.  pertussis. 

B.  pertussis,  like  the  influenza  bacillus,  is  an  aerobic  organism. 
Anaerobic  development  has  not  been  obtained.  The  optimum  tem- 
perature of  growth  is  37°  C.;  Wollstein3  states  that  slight  development 
takes  place  even  at  5°  to  10°  C.  An  exposure  of  thirty  minutes  at 
57°  to  60°  C.  prevents  further  development  in  artificial  media.  The 
organisms  may  remain  viable  upon  the  potato-glycerin-agar  medium 
for  two  months. 

Products  of  Growth. — According  to  Wollstein,4  no  acid  is  produced 
in  dextrose,  lactose,  saccharose  or  mannite  serum  broth.  No  enzymes 
have  been  demonstrated  in  cultures  of  the  organism,  and  it  produces 
no  visible  changes  in  hemoglobin.  Extracellular  toxins  have  never 
been  demonstrated,  but  autolyzed  cultures  introduced  intravenously 

1  It  is  prepared  as  follows:    100  grams  finely  chopped  potatoes  are  boiled  in  200  c.c. 
of  4  per  cent,  glycerin  for  a  short  time,  then  cooled.     To  every  100  c.c.  of  the  potato 
glycerin  extract  there  is  added  300  c.c.  of  7.5  per  cent,  agar  containing  0.5  per  cent. 
NaCl.     The  glycerin  potato  extract  replaces  the  usual  peptone-meat-juice  nutrients  of 
nutrient  agar.     The  mixture  is  heated  to  boiling,  filtered,  and  sterilized  in  test-tubes 
about  3  c.c.  of  medium  per  tube.     (Old  potatoes  which  are  slightly  alkaline  in  reaction 
are  much  better  than  new  potatoes  which  are  usually  acid  in  reaction,  in  preparing  the 
glycerin  extract.)     To  each  tube  of  the  sterilized  glycerin  potato  agar  medium  is  added 
an  equal  volume  of  sterile,  defibrinated  human  or  rabbit's  blood,  while  the  medium  is 
still  warm,  45°  to  50°  C.     Then  the  mixture  is  cooled  in  an  inclined  position. 

2  Wollstein,  Jour.  Exp.  Med.,  1909,  xi,  41. 

3Loc.  cit.  4  LOC.  cit. 


422  HEMOGLOBINOPHILIC  BACILLI 

into  rabbits  frequently  kill  them  within  twenty-four  to  forty-eight 
hours.  Subcutaneous  injections  of  autolysates  may  cause  local 
necrosis,  but  generalized  symptoms  fail  to  appear.  Similar  results 
have  been  obtained  with  endotoxin  obtained  by  grinding  the  bacilli 
to  an  impalpable  powder  and  injecting  a  saline  suspension  of  it.1 

Pathogenesis. — Animal. — Klimenko2  and  Franker3  produced  a  catarrh 
of  the  respiratory  mucosa  of  monkeys  and  young  dogs  by  intratracheal 
injections  of  B.  pertussis  suspended  in  salt  solution.  A  febrile  reac- 
tion appeared  after  three  to  four  days  and  several  of  the  animals  died 
within  two  to  three  weeks.  The  bacilli  were  recovered  from  the 
bronchial  mucus,  bronchi,  and  from  the  areas  of  bronchopneumonia 
which  developed  in  the  lungs.  No  characteristic  paroxysms  were 
induced,  although  Klimenko  stated  that  sneezing  and  coughing  were 
noticed.  Wollstein4  has  pointed  out  a  possible  source  of  error  in  the 
dog  experiments:  she  finds  that  those  dogs  which  die  after  injections 
of  B.  pertussis  succumb  to  canine  distemper;  the  lesions  of  the  respira- 
tory tract  are  readily  accounted  for  on  this  basis,  and  the  blood  of 
the  animals  fails  to  react  specifically  with  the  Bordet-Gengou  bacillus. 

Human. — There  are  no  postmortem  lesions  characteristic  of  whoop- 
ing-cough. Bronchopneumonia  is  the  most  common  complication 
seen  at  autopsy.  Mallory  and  Hornor,5  and  Mallory,  Hornor  and 
Henderson6  have  advanced  an  interesting  explanation  for  the  parox- 
ysms of  whooping-cough.  They  find  that  the  ciliated  epithelium  of 
the  respiratory  tract  is  denuded  in  places  and  the  cilia  plastered  down 
to  such  an  extent  as  to  interfere  with  the  free  removal  of  mucus  by 
the  mechanical  action  of  the  bacteria.  When  mucus  accumulates  in 
sufficient  amount,  jt  is  forcibly  expelled  by  a  prolonged  violent  parox- 
ysm of  coughing.  These  experiments  were  made  upon  animals;  the 
frequent  occurrence  of  B.  bronchosepticus  or  a  closely-related  bacillus 
in  the  respiratory  tracts  of  laboratory  animals,  which  produces  similar 
lesions  to  those  seen  in  canine  distemper,  should  be  borne  in  mind  in 
interpreting  these  results. 

Immunity. — Whooping-cough  is  more  commonly  a  disease  of  chil- 
dren, and  recovery  from  one  attack  appears  to  confer  lifelong  immunity 
as  a  rule. 


1  Bordet  and  Gengou,  Centralbl.  f.  Bakt.,  Ref.,  1909,  xliii,  273. 

2  Centralbl.  f.  Bakt.,  Orig.,  1909,  xlviii,  64. 

3  Munch,  med.  Wchnschr.,  1908,  p.  1683. 

4  Loc.  cit. 

6  Jour.  Med.  Research,  1912,  xxvii,  115. 
6  Ibid.,  1913,  xxvii,  No.  4. 


THE  KOCH-WEEKS  BACILLUS  423 

Bacteriological  Diagnosis. — 1.  Morphological. — The  diagnosis  of 
whooping-cough  by  a  microscopical  examination  of  bronchial  dis- 
charges is  not  satisfactory.  Influenza  bacilli  are  frequently  present 
in  the  mucus  and  sputum  from  cases  of  pertussis,  and  no  method  is 
available  at  the  present  time  which  will  distinguish  with  certainty 
between  the  two  bacilli. 

2.  Cultural. — The  isolation  of  B.  pertussis  from  bronchial  mucus 
upon  potato-glycerin-blood  agar  and  its  ability  to  grow  upon  ascitic 
media  free  from  hemoglobin  separates  the  Bordet-Gengou  organism 
from  B.  influenzas 

3.  Serological. — The  sera  of  animals  highly  immunized  to  B.  per- 
tussis agglutinate  the  organism  in  high  dilution,  but  fail  to   agglu- 
tinate B.  influenzse  and  vice  versa.    The  serum  of  patients  during  and 
after  recovery  from  whooping-cough,  however,  agglutinates  B.  per- 
tussis irregularly,  and  the  method  has  no  general  diagnostic  impor- 
tance.   The  method  of  complement  fixation  similarly  has  not  been 
successful  as  applied  to  the  diagnosis  of  the  disease  in  man,  although 
the  reaction  is  clear-cut  when  applied  to  the   sera  of  immunized 
animals.1 

The  etiology  of  whooping-cough  has  not  been  definitely  established; 
the  Bordet-Gengou  bacillus,  however,  is  found  in  the  majority  of  cases 
of  pertussis.  Up  to  the  present  time  it  has  not  been  isolated  from 
healthy  subjects. 

THE   KOCH-WEEKS   BACILLUS. 

Acute  contagious  conjunctivitis  or,  as  it  is  popularly  known, 
pink-eye  is  generally  considered  to  be  an  infection  of  the  conjunctiva 
by  a  small  bacillus  first  described  by  Koch.2  Somewhat  later  Weeks3 
described  the  organism  anew  and  succeeded  in  growing  it  in  artificial 
media,  probably  in  association  with  other  organisms.  Kartulis4 
isolated  it  in  pure  culture  on  blood  serum,  and  Kamen5  published  a 
more  complete  study  of  the  cultural  characters  of  the  organism. 

Morphology. — The  Koch-Weeks  bacillus  is  a  small  rod-shaped 
organism  resembling  the  influenza  bacillus.  It  is  of  about  the  same 
diameter  as  the  influenza  bacillus,  0.25  micron,  but  somewhat  longer, 
measuring  from  1  to  2  microns  in  length.  It  occurs  singly  and  in 

1  Wollstein,  loc.  cit. 

2  Wien.  klin.  Wchnschr.,  1883,  1550;    Arb.  a.  d.  kais.  Gesamte.,  1887,  iii,  19. 

3  Arch,  of  Ophthalmology,  1886,  xv,  No.  4. 

4  Centralbl.  f.  Bakt.,  1899,  i,  449. 
'  Ibid.,  1889,  xxv,  401,  449. 


424  HEMOGLOBINOPHILIC  BACILLI 

pairs,  but  short  chains  of  bacilli  are  not  uncommonly  seen  in  growths 
on  artificial  media.  Involution  forms,  which  are  atypical  in  form  and 
size,  are  also  found  in  cultures  outside  of  the  body.  The  organism  is 
non-motile  and  it  has  no  flagella.  Capsules  and  spores-  have  not 
been  demonstrated.  The  Koch-Weeks  bacillus  stains  with  ordinary 
anilin  dyes,  but  not  intensely.  It  is  Gram-negative. 

Isolation  and  Culture. — The  organism  grows  best  in  a  medium  of 
semi-liquid  consistency.  5  per  cent,  agar  containing  blood  or  ascitic 
fluid  appears  to  be  the  best  for  this  purpose.  Material  for  inoculation 
is  conveniently  obtained  first  by  flushing  the  conjunctiva  thoroughly 
with  sterile  salt  solution  then  removing  some  of  the  secretion  which 
soon  accumulates  with  a  sterile  swab  which  is  immediately  rubbed 
upon  the  surface  of  the  blood  agar.  After  twenty-four  to  forty-eight 
hours'  incubation  at  37°  C.  colonies  usually  appear  which  are  very 
minute  and  colorless.  They  die  rapidly. 

Resistance. — The  Koch-Weeks  bacillus  is  very  susceptible  to  drying 
and  to  heat;  chemical  disinfectants  very  rapidly  destroy  the  organism 
outside  the  human  body. 

Nothing  is  known  of  the  products  of  growth. 

Pathogenesis. — Attempts  to  produce  conjunctivitis  in  animals  with 
the  organism  have  been  uniformly  negative  but  inoculations  upon 
the  healthy  conjunctiva  of  man  usually  reproduce  the  disease. 

The  disease  is  very  contagious;  it  is  spread  chiefly  by  contact. 

MORAX-AXENFELD  BACILLUS. 

In  1896  Morax1  described  a  diplobacillus  which  he  observed  repeat- 
edly during  an  epidemic  -of  subacute  conjunctivitis.  The  year  follow- 
ing, Axenfeld2  published  an  excellent  description  of  the  same  organism, 
which  is  commonly  referred  to  as  the  Morax-Axenfeld  bacillus  or  the 
diplobacillus  of  subacute  conjunctivitis. 

Morphology. — The  organisms,  as  the  name  implies,  occur  typically 
in  pairs;  less  frequently  they  may  remain  adherent  to  form  short 
chains.  The  individual  bacilli  are  of  average  size,  measuring  from 
1  to  2  microns  in  length,  and  about  1  micron  in  diameter  as  an  average. 
The  ends  of  the  bacilli  are  rather  square  cut.  Cultures  on  artificial 
media  are  somewhat  variable  in  size  and  shape;  chain  formation  is  not 
uncommon  and  involution  forms  are  frequent.  The  organisms  are 
non-motile  and  possess  no  flagella.  Neither  spores  nor  capsules  have 

1  Ann.  Inst.  Past.,  June,  1896.  2  Centralbl.  f.  Bakt.,  1897,  xxi,  1. 


BACILLUS  OF  DUCREY  425 

been  demonstrated.  Ordinary  anilin  dyes  color  the  bacilli  readily, 
and  the  Gram  stain  is  negative. 

Isolation  and  Culture. — Growth  occurs  only  in  media  containing 
blood  serum  or  ascitic  fluid;  Loffler's  alkaline  blood  serum  is  a  favorable 
substrate.  The  colonies  on  blood  serum  after  twenty-four  to  forty- 
eight  hours'  development  at  37°  C.  are  slightly  sunken,  due  to  the 
liquefaction  of  the  medium.  After  several  days  the  serum  is  almost 
completely  liquefied.  Colonies  grown  on  ascitic  agar  are  small,  color- 
less and  transparent  even  after  several  days'  incubation.  Oxygen 
is  essential  for  the  growth  of  the  bacilli;  no  growth  occurs  when 
oxygen  is  excluded  from  the  media. 

Prognosis  of  Growth. — A  proteolytic  enzyme  which  liquefies  coagu- 
lated blood  serum  is  the  only  enzyme  which  has  been  described.  No 
other  products  of  growth  are  known. 

Pathogenesis. — Attempts  to  reproduce  the  characteristic  subacute 
conjunctivitis  in  experimental  animals  have  utterly  failed.  In  man 
the  typical  disease  is  a  subacute  catarrhal  conjunctivitis  with  com- 
paratively little  pus  formation,  differing  in  this  respect  sharply  from 
the  acute  conjunctivitis  which  is  produced  by  the  Koch-Weeks  bacil- 
lus. The  angles  of  the  eye  are  inflamed,  particularly  the  caruncles. 
The  organisms  are  best  detected  in  the  secretion  which  collects  during 
the  night.  They  occur  both  in  pus  cells  and  free,  frequently  in  con- 
siderable numbers.  Morax  appears  to  have  reproduced  the  essential 
lesions  by  inoculating  a  drop  of  culture  upon  a  healthy  conjunctiva. 

BACILLUS   OF   DUCREY. 

The  soft  chancre,  chancroid,  or  soft  sore  must  be  sharply  differen- 
tiated from  the  hard  chancre  with  which  it  has  nothing  in  common. 
The  soft  chancre  is  a  non-specific,  ulcerating  sore  common  to  both 
sexes,  particularly  among  the  unclean.  It  begins  as  a  small  red  spot 
which  rapidly  develops  into  a  pustule.  This  pustule  soon  breaks  down, 
leaving  a  spreading  ulcer  in  which  necrosis  is  a  prominent  feature. 
The  ulcer  spreads  with  considerable  rapidity  and  is  difficult  to  control. 
The  adjacent  and  regional  lymph  glands  usually  become  involved 
and  they  soon  soften  and  ulcerate. 

Ducrey1  first  called  attention  to  a  bacillus  (which  bears  his  name), 
which  he  found  regularly  in  chancroids.  In  1900  Besancon,  Griffon 
and  Le  Sourd2  succeeded  in  growing  the  organism  in  pure  culture  upon 

1  Monatsch.  f.  prakt.  Dermat.,  1889,  ix,  No.  9. 

2  Gaz.  dcs  Hop.,  1900,  No.  14;   Ann.  de  Dermat.  et  Syph.,  1901,  ii,  1. 


426  HEMOGLOBINOPHILIC  BACILLI 

blood  agar.  The  organism  is  often  referred  to  as  the  Streptobacillus 
of  Ducrey. 

Morphology. — The  bacillus  of  Ducrey  is  a  small  bacillus,  measuring 
about  0.5  micron  in  diameter  and  from  1  to  2  microns  in  length.  It 
occurs  characteristically  both  in  chancroids  and  in  culture  in  chains 
of  considerable  length.  Frequently  these  streptobacilli  are  found  in 
dense  masses.  The  organism  stains  with  ordinary  anilin  dyes;  usually 
the  stain  is  more  intense  at  the  ends  of  the  rod,  the  centre  being  nearly 
devoid  of  color.  This  gives  the  organism  a  diplococcoid  appearance. 
It  is  Gram-negative. 

Isolation  and  Culture. — The  Ducrey  bacillus  does  not  grow  upon 
ordinary  media,  but  cultures  may  be  obtained  by  the  method  of 
Davis,1  which  consists  essentially  in  sterilizing  the  skin  over  an 
unbroken  bubo  and  aspirating  the  contents  with  a  hypodermic  needle 
which  has  been  maintained  at  body  temperature.2  The  material  is 
introduced  directly  upon  the  surface  of  blood  agar.3  If  the  ulcers  or 
buboes  have  opened,  they  may  be  cleaned  with  sterile  gauze,  dried, 
then  painted  with  tincture  of  iodin  and  covered  with  sterile  gauze. 
Inoculations  upon  blood  agar  are  made  from  the  pus  which  collects 
under  the  gauze  within  twenty-four  hours. 

The  colonies  are  usually  visible  after  twenty-four  hours'  incubation 
at  37°  C.  They  appear  as  raised,  shining,  grayish  droplets  with  a 
pearly  lustre.  They  die  out  rapidly  at  room  temperature,  but  may  be 
kept  alive  at  body  temperature  for  some  days.  The  colonies  are 
removed  from  the  medium  with  some  difficulty,  for  they  tend  to  slip 
away  from  the  platinum  needle.  Subcultures  tend  to  increase  some- 
what in  luxuriance  of  growth,  and  by  frequent  transfer  the  organism 
may  be  kept  alive  for  weeks  provided  the  growths  are  maintained  at 
37°  C. 

The  bacillus  of  Ducrey  is  an  aerobic  organism  which  is  not  resistant 
to  drying.  The  pus  becomes  non-infective  after  one  or  two  days' 
desiccation.  Weak  antiseptics  quickly  destroy  it. 

The  products  of  growth  are  unknown. 

Pathogenesis. — It  is  non-pathogenic  for  ordinary  laboratory  animals. 
Tomasczewski4  claims  to  have  reproduced  a  chancroid  in  a  monkey 

1  Jour.  Med.  Research,  1893,  ix,  401. 

2  Both  the  syringe  and  the  blood  agar  should  be  warmed  to  body  temperature  before 
use,  because  the  organism  rapidly  loses  its  viability  at  room  temperature. 

3  Blood  agar  for  this  purpose  is  prepared  by  adding  defibrinated  human  or  rabbit 
blood  to  agar;  the  medium  is  heated  to  56°-60°  C.  for  thirty  minutes  to  destroy  natural 
bactericidal  substances,  and  incubated  for  twenty-four  hours  to  insure  sterility. 

4  Deutsch.  med.  Wchnschr.,  1903,  No.  26. 


BACILLUS  OF  DUCREY  427 

(Macacus)  by  the  injection  of  a  blood  agar  culture  obtained  from  a 
bubo.  This  same  culture  also  produced  a  chancroid  when  inoculated 
into  a  man.  Several  successful  inoculations  in  a  man  are  recorded 
which  appear  to  establish  satisfactorily  the  etiological  relationship 
of  the  bacillus  of  Ducrey  to  the  lesion. 

Bacteriological  Diagnosis. — 1.  Microscopical. — If  material  be  removed 
carefully  from  the  base  of  an  ulcer  and  spread  gently  upon  a  glass 
slide  to  prevent  the  breaking  up  of  the  characteristic  arrangement  of 
the  bacilli  in  long  intertwined  chains,  a  definite  diagnosis  may  fre- 
quently be  made  by  direct  observation  of  the  Gram-stained  prepara- 
tion under  the  microscope. 

2.  Cultural. — Material  preferably  obtained  from  an  unopened  bubo 
should  be  spread  upon  the  surface  of  blood  agar,  employing  the  technic 
outlined  above.    As  much  material  as  possible  should  be  inoculated 
to  insure  growth  of  the  bacilli. 

3.  Inoculation  of  Patient. — The  forearm  of  the  patient  is  thoroughly 
cleaned,  then  scarified  with  a  platinum  needle  infected  with  material 
from  the  ulcer  or  from  a  pure  culture.     The  lesion  appears  within 
twenty-four  hours  and  it  is  typically  developed  in  from  three  to  five 
days.    It  is  obvious  that  little  or  no  immunity  is  produced,  because 
autoinoculation    results    in    infection.      The    possibility    of    syphilis 
must  be  borne  in  mind  in  inoculation  experiments,  particularly  in 
transferring  material  from  one  subject  into  another.     Syphilis  and 
chancroid  may  exist  in  the  same  patient. 


CHAPTER  XXIII. 

THE  TUBERCLE  BACILLUS  GROUP:    HUMAN,  BOVINE, 

AND  AVIAN. 

THE   ACID-FAST   GROUP. 

THERE  is  a  well-defined  group  of  bacteria  characterized  by  the 
relatively  large  amounts  of  lipoidal  substances  contained  within  their 
bodies.  These  lipoidal  or  waxy  substances  confer  upon  the  members 
of  the  group  distinctive  staining  reactions;  ordinary  dyes  do  not 
stain  them  at  all,  or  at  best  slowly.  The  more  intense  stains  con- 
taining a  mordant,  as  carbol-fuchsin,  penetrate  the  waxy  envelope, 
especially  when  heat  is  applied;  once  stained  the  bacteria  retain 
the  dye  tenaciously  even  after  treatment  with  mineral  acids.  This 
resistance  to  decolorization  with  acids  has  led  to  the  name — the 
Acid-fast  group. 

Included  within  the  group  of  acid-fast  bacteria  are  saprophytic 
types  found  rather  commonly  in  hay  and  manure;  parasitic  organ- 
isms found  upon  the  surface  of  the  body,  as  the  smegma  bacillus  and 
the  nasal  secretion  bacillus  of  Karlinski;  and  exquisitely  pathogenic 
bacteria,  Bacillus  tuberculosis  and  Bacillus  leprse.  The  basis  o* 
classification  therefore  is  chemical  rather  than  morphological,  and  in 
this  sense  the  definition  of  the  acid-fast  organisms  does  not  follow  a 
strictly  natural  system. 

Types  of  Tubercle  Bacilli. — Four  types  of  tubercle  bacilli  are 
recognized  which  are  pathogenic  respectively  for  man,  cattle,  birds, 
and  for  fishes  and  reptiles;  the  human,  bovine,  avian,  and  ichthic 
varieties.  Considerable  discussion  has  arisen  concerning  the  identity 
of  the  human,  bovine,  and  avian  varieties,  some  authorities  claiming 
that  they  are  identical,  although  modified  somewhat  by  their  con- 
tinuous sojourn  in  a  series  of  animals  of  the  same  kind.  The  evidence 
for  this  view  is  arrayed  around  the  observation  that  tubercle  bacilli 
of  undoubted  bovine  type  occasionally  are  isolated  from  tuberculous 
lesions  in  man  (chiefly  in  children,  infrequently  in  adults).  On  the 
other  hand  human  bacilli  are  less  commonly  found  in  progressive 
tuberculous  lesions  of  cattle.  In  spite  of  many  attempts  to  change 


PLATE  II 


Tubercle  Bacilli;  Ziehl-Neelsen  Stain. 


TUBERCLE  BACILLUS  429 

one  type  into  the  other,  no  experiments  have  been  reported  up  to  the 
present  time  which  are  sufficiently  conclusive  and  extensive  to  war- 
rant the  assumption  that  one  variety  has  been  permanently  changed 
into  the  other.  Loss  or  increase  of  pathogenic  properties  of  one 
strain  does  not  suffice  to  bridge  the  gap  between  it  and  another  strain 
habitually  pathogenic  for  another  animal.  It  is  very  probable  that 
the  human,  bovine,  and  avian  strains  had  a  common  ancestor  and 
that  acclimatization  in  different  animals  has  led  to  the  perpetuation 
of  three  culturally  and  pathogenically  stable  varieties.  The  ichthic 
type  is  much  more  closely  related  to  the  non-pathogenic  grass  and 
dung  bacilli  than  to  the  true  tubercle  bacilli. 

TUBERCLE   BACILLUS. 

Historical. — One  of  the  greatest  chapters  in  the  history  of  medicine 
was  inaugurated  by  the  isolation  of  the  tubercle  bacillus  in  pure  cul- 
ture, and  the  demonstration  of  its  etiological  relationship  to  tuber- 
culosis. The  credit  for  this  work,  which  in  every  detail  marks  an 
important  epoch  in  bacteriology,  belongs  to  Robert  Koch.1 

Morphology. — The  tubercle  bacillus  is  a  slender,  straight  or  slightly 
curved  rod  measuring  from  0.2  to  0.6  micron  in  diameter  and  from 
1.5  to  6  microns  in  length.  The  size  and  appearance  of  the  organism 
varies  somewhat  according  to  the  source.  In  sputum  it  frequently 
occurs  in  small  clumps,  often  with  the  long  axes  of  the  bacilli  parallel. 
Occasionally  a  pair  of  bacilli  are  arranged  at  an  angle  like  the  letter 
"V."  The  bacilli  are  typically  isodiametric,  but  irregularities  of 
outline  are  not  uncommon;  these  irregularities  are  due  to  nodules 
which  cause  the  organism  to  swell  or  bulge  wherever  they  occur. 
These  nodules  frequently  stain  deeply,  and  between  them  are  areas 
which  stain  lightly  or  not  at  all,  thus  giving  the  rod  a  beaded  or  vacuo- 
lated  appearance  which  may  be  so  marked  that  the  organism  resembles 
a  chain  of  cocci.  These  "beaded"  forms  are  frequently  observed  in 
the  sputum  of  consumptives  and  occasionally  in  old  growths  on 
artificial  media. 

True  branching  is  also  occasionally  exhibited  by  tubercle  bacilli 
derived  both  from  the  sputum  and  from  culture.2  Some  observers 
have  classed  the  tubercle  bacillus  with  the  group  of  Actinomyces  on 
the  basis  of  this  branching.3 

1  Berl.  klin.  Woch.,  1882,  No.  15;  Mitt.  a.  d.  Kais.  Ges.-Amte,  1884,  ii,  1. 

2  Klein,  Centralbl.  f.  Bakt.,  1890,  vii,  794. 

3  Babes  and  Levaditi,  Arch,  de  med.  exper.  et  d'anat.  path.,  1897,  ix,  1041. 


430  THE   TUBERCLE  BACILLUS  GROUP 

The  tubercle  bacillus  is  non-motile  and  possesses  no  flagella.  It 
forms  no  capsule  but  possesses  a  waxy  envelope  which  confers  upon 
the  organism  unusual  resistance  to  desiccation  and  to  the  action  of 
chemicals.  No  spores  have  been  definitely  demonstrated,  but  Koch1 
believed  that  the  deeply  staining  granules  found  in  the  bacillus  might 
be  true  endospores.  The  generally  accepted  view  is  opposed  to  this 
supposition.2 

Staining. — Tubercle  bacilli  and  closely  related  organisms  possess 
in  common  a  relatively  large  amount  of  waxy  substance3  which  is 
relatively  impervious  even  to  the  more  intense  stains,  as  carbol-fuchsin. 
Ordinary  anilin  dyes  do  not  stain  members  of  the  tubercle  bacillus 


FIG,  61. — Tubercle  bacilli,  beaded  forms. 

group.  They  are  Gram-positive,  but  it  requires  several  hours  for  the 
anilin-oil  gentian  violet  to  color  the  organisms.  When  a  stain  has 
penetrated  the  substance  of  the  tubercle  bacillus  it  is  retained  with 
great  tenacity;  alcohol  and  even  rnineral  acids  in  moderate  concen- 
tration fail  to  remove  it  except  after  long  exposure.  The  members 
of  the  tubercle  bacillus  group  vary  somewhat  in  this  resistance  to 
decolorization;  the  true  tubercle  bacilli  are  both  "alcohol-"  and 
"acid-fast;"  other  organisms  in  the  group  may  be  either  "alcohol-" 
or  "acid-fast."  Young  tubercle  bacilli  are  frequently  non-acid-fast.4 

1  Mitt.  a.  d.  Kais.  Gesamte,  1884,  ii,  22. 

2  See  Wherry,  Centralbl.  f.  Bakt.,  Orig.,  1913,  Ixx,  Heft  3-4.    Conditions  which  favor 
the  formation  of  "spores"  in  certain  acid-fast  bacteria. 

3  For    chemical    composition    of    fatty  substance    of   the    tubercle    bacillus   see:   de 
Schweinitz  and  Dorset,  Jour.  Am.  Chem.    Soc.,    1898,  xx,  No.  8,  p.  618;  20th  Annual 
Report,  Bur.   Animal   Ind.,    1903.     Levene,   Med.   Record,   December   17,    1898,  873; 
Jour.  Med.  Research,  1901,  vi,  120;  1904,  xii,  251.     Kresling,  Centralbl.  f.  Bakt.,  1901, 
xxx,  897. 

4  Wolbach  and  Ernst,  Jour.  Med.  Research,  1903,  x,  No,  3. 


TUBERCLE  BACILLUS 


431 


The  best  and  most  universally  applicable  stain  for  the  tubercle 
bacillus  is  the  Ziehl-Neelsen  stain.1    It  is  used  as  follows: 

1.  A  thin  smear  of  the  material  to  be  examined  for  tubercle  bacilli 
is  prepared  and  fixed  in  the  usual  manner,  then  flooded  with  carbol- 
fuchsin  and  steamed  gently  (not  boiled)  for  five  minutes.    The  pre- 
paration must  be  flooded  continuously  with  the  stain. 

2.  Wash  thoroughly  with  water  to  remove  the  excess  of  stain. 

3.  Decolorize  with  90  per  cent,   alcohol   containing   3   per   cent, 
hydrochloric  acid  until  the  pink  color  has  practically  disappeared. 

4.  Wash  with  water. 

5.  Counterstain  lightly  with  Loffler's  alkaline  methylene  blue. 

6.  Wash,  dry,  examine. 


*  'l 


FIG.  62.— Tubercle  bacillus  showing  branching.      X  1800.     (Wolbach  and  Ernst.) 

Tubercle  bacilli  are  colored  red;  non-acid-fast  bacteria  are  colored 
blue.  It  should  be  remembered  that  spores  may  also  be  stained  red 
by  this  method,  but  they  are  not  likely  to  be  confused  with  tubercle 
bacilli;  they  are  round  or  oval;  tubercle  bacilli  are  much  longer. 

The  decolorization  and  counterstaining  may  be  accomplished  by 
one  operation,  according  to  the  Frankel-Gabbett  method.2  The 
preparation  of  the  smear  and  staining  with  carbol-fuchsin  is  carried 
out  as  above  (Steps  1  and  2).  Decolorization  and  counterstaining  are 
accomplished  by  flooding  the  preparation  with  the  Frankel-Gabbett 
solution  (100  c.c.  water,  25  c.c.  sulphuric  acid,  50  c.c.  saturated  alco- 
holic solution  of  methylene  blue)  for  three  to  five  minutes,  then  wash 


1  Ziehl,  Deutsch.  med.  Wchnschr.,  1882,  451;    Neelsen,  Fortschr.  d.  Med.,  1885,  200. 
'Frankel,  Beil.  klin,  Woch.,  1884;  Gabbett,  Lancet,  1887. 


432  THE   TUBERCLE  BACILLUS  GROUP 

with  water,  dry  and  examine.  Acid-fast  bacteria  are  stained  red, 
all  other  organisms  are  blue. 

Much  Granules. — Certain  granules  are  found  in  old  caseous  foci 
and  occasionally  in  the  pus  of  cold  abscesses  which  do  not  contain 
tubercle  bacilli  demonstrable  by  the  acid-fast  stain.  Material  con- 
taining these  granules  introduced  into  guinea-pigs  causes  a  rapidly 
fatal  tuberculosis.  Much1  states  that  these  granules  are  living  frag- 
ments of  tubercle  bacilli  which  develop  into  the  typical  bacillus  when 
environmental  conditions  are  optimum.  They  are  Gram-positive  and 
non-acid-fast,  but  may  regain  their  acid-fastness.  When  they  are 
non-acid-fast  they  do  not  multiply.  The  exact  significance  of  these 
granules  (Much  granules)  is  as  yet  undetermined;  whether  they  are 
identical  with  the  "splinters"  described  by  Spengler2  is  problematical. 
The  "splinters"  are  usually  colored  red  with  fuchsin,  and  they  frequently 
appear  in  tubercle  bacilli  that  do  not  stain  uniformly,  appearing  as 
rows  of  red,  acid-fast  granules.  According  to  Spengler  they  may  be 
found  in  sputum  or  other  tuberculous  material  as  heaps  of  small 
granules,  even  if  the  bacilli  themselves  cannot  be  demonstrated. 

Isolation  and  Culture. — It  is  difficult  to  cultivate  the  tubercle  bacillus 
directly  from  lesions  upon  artificial  media  and  it  is  even  more  diffi- 
cult to  obtain  pure  cultures  directly  from  sputum,  feces,  or  lung 
cavities  where  tubercle  bacilli  are  growing  in  the  presence  of  other 
organisms  which  develop  much  more  rapidly  on  artificial  media.  The 
initial  growth  on  artificial  media  is  the  most  difficult  to  obtain.  Either 
coagulated  dog's  serum3  or  the  Dorset  egg  medium4  is  best  for  this 
purpose.  Tissue  containing  tubercles  is  removed  from  the  animal 
with  aseptic  precautions  to  sterile  Petri  dishes.  The  tissue  is  minced 
somewhat  and  then  distributed  over  the  slanted  surface  of  either  the 
serum  or  the  egg  medium.  At  the  end  of  a  week  or  ten  days  the  bits 
of  tissue  are  moved  around  to  fresh  surface  areas;  at  the  end  of  two 
to  four  weeks  the  tubercle  bacilli  appear  as  minute  gray  nodules 
which  gradually  spread,  forming  eventually  a  wrinkled  dull  gray- 
yellow  growth  covering  the  greater  part  of  the  medium.  Subcul- 
tures from  the  original  culture  grow  better  on  artificial  media  than 
the  original  culture,  although  even  subcultures  grow  very  slowly. 

1  Beitr.  z.  Klinik  d.  Tuberkulose,  1907,  viii,  85,  357,  368;    1908,  xi,  67;    1913,  Supp. 
Bd.  vi. 

2  Deutsch.  med.  Wchnschr.,  1907,  p.  337. 

3  Coagulated  at  75°  C.;  Theobald  Smith,  Jour.  Exp.   Med,   1898,  iii.  647;  Trans. 
Assn.  Am.  Phys.,  1898,  xiii,  417. 

4  Bureau  of  Animal  Industry.  Annual  Report,  1902,  p.  574. 


TUBERCLE  BACILLUS  433 

The  coagulated  serum  or  the  egg  medium  may  be  used  for  subcultures ; 
glycerin  agar  or  glycerin  potato  is  also  suitable  for  this  purpose.  It 
is  essential  to  protect  the  cultures  from  evaporation  and  to  incubate 
them  in  a  slanting  position.  This  is  best  accomplished  by  sealing  the 
slant  cultures  after  they  are  made,  either  with  paraffin  or  with  corks 
which  have  been  charred  to  kill  off  moulds  or  other  organisms,  then 
covered  with  lead  foil.  Tubercle  bacilli  grow  fairly  readily  on  the 
surface  of  glycerin  broth  after  they  have  become  accustomed  to 
artificial  media.  A  fresh  thin  film  from  egg  medium  floated  on  the 
surface  of  the  broth  is  the  best  method  of  obtaining  the  growth  in 
this  medium.  The  organisms  must  be  floated  on  the  surface  of  the 
broth,  otherwise  growth  does  not  take  place.  If  the  growth  sinks  to 
the  bottom  all  development  ceases.  Tubercle  bacilli  do  not  grow 
readily  in  gelatin  or  other  artificial  media  not  containing  glycerin 
or  proteins  derived  from  blood  serum  or  egg. 

Cultures  of  tubercle  bacilli  which  have  been  grown  on  artificial 
media  for  some  time  may  be  gradually  accustomed  to  develop  in  media 
of  simple  composition.  Proskauer  and  Beck1  grew  the  organism  upon 
the  Uschinsky  medium  to  which  glycerin  was  added:  Wherry2  and 
Lowenstein3  have  employed  media  in  which  ammonium  salts  were 
the  only  source  of  nitrogen.  Kendall,  Day  and  Walker4  have  corro- 
borated these  results.  Tuberculin  appears  to  be  produced  even  in 
these  simple  media.  The  tubercle  bacillus  grows  in  milk,  producing 
a  gradual  solution  of  the  casein.5 

The  tubercle  bacillus  is  aerobic,  although  it  will  develop  slowly 
anaerobically.  Its  temperature  range  is  rather  limited,  the  organisms 
growing  between  30°  C.  and  42°  C.,  with  an  optimum  temperature 
of  37°  C.  Growth  below  35°  C.  is  slow.  Tubercle  bacilli  are  fairly 
resistant  to  drying,  naked  germs  being  killed  by  dry  heat  at  100°  C. 
only  after  forty-five  minutes.  With  moist  heat  an  exposure  to  60° 
C.  kills  them  in  thirty  minutes,  65°  C.  in  fifteen  minutes,  70°  C.  in 
five  minutes,  80°  C.  in  one  minute,  and  100°  C.  in  half  a  minute. 
The  organisms  enclosed  in  mucus  are  much  more  resistant,  dry  heat 
(100°  C.)  killing  them  only  after  an  exposure  of  from  two  to  three 

1  Ztschr.  f.  Hyg.,  1894,  xviii,  128. 

2  Jour.  Inf.  Dis.,  1913,  xiii,  144;  Centralbl.  f.  Bact.,  Orig.,  1913,  Ixx,  115. 

3  Centralbl.  f.  Bakt.,  Orig.,  1913,  Ixviii,  591. 

4  Jour.  Inf.  Dis.,  1914,  xv,  428. 

6  Klein,  Centralbl.  f.  Bakt.,  Orig.,  1900,  xxviii,  111.     Monvoisin,  Compt.  rend.  Acad. 
Sci.,  October,  1909,  xxvi;    Rev.  de  Med.  vcterin.,  1910,  Ixxxvii,  16.     Mossu  and  Mon- 
voisin, Compt.  rend.  Soc.  Biol.,  1907,  Uii,  No.  26.     Kendall,  Day  and  Walker,  Jour, 
Am.  Chem.  Assn.,  1914,  xxvi,  1959. 
28 


434  THE  TUBERCLE  BACILLUS  GROUP 

hours,  70°  C.  after  seven  hours,  and  60°  C.  after  ten  hours.  In  sterile 
water  the  organisms  may  remain  alive  for  over  two  months.  They 
are  quite  resistant  also  to  putrefaction.  Instances  are  on  record 
where  tuberculous  lungs  have  been  buried  for  six  months  and  yet 
contained  virulent  organisms.  Schottelius  claims  that  a  tuberculous 
lung  buried  two  years  contained  virulent  tubercle  bacilli  at  the  end 
of  that  time. 

The  thermal  death  point  in  milk  is  60°  C.  for  thirty  minutes.  There 
is  d  source  of  error  in  determining  the  thermal  death  point  of  the 
tubercle  bacillus  or  of  any  other  organism  in  milk.  If  the  experiment 
is  carried  out  in  milk  which  is  not  enclosed  in  such  a  manner  as  to 
prevent  surface  evaporation  the  results  are  inaccurate;  the  scum 
which  forms  on  the  surface  of  the  milk  as  the  result  of  evaporation 
contains  casein  and  salts;  they  are  non-conductors  of  heat  and  protect 
the  organisms  so  that  they  apparently  resist  a  much  higher  tempera- 
ture than  would  otherwise  be  the  case.1 

Tubercle  bacilli  in  sputum  are  killed  in  twenty-four  hours  by  mixing 
the  sputum  with  an  equal  volume  of  5  per  cent,  carbolic  acid.  Mer- 
curic chloride  is  not  suitable  for  this  purpose  because  it  precipitates 
mucus,  forming  a  compound  with  it  which  renders  its  germicidal 
action  nil.  Rooms  containing  tubercle  bacilli  may  be  disinfected  either 
by  burning  four  pounds  of  sulphur  to  1000  cubic  feet  in  a  moist  atmos- 
phere, or  by  evaporating  500  c.c.  of  formaldehyde  to  every  1000  cubic 
feet  under  the  same  conditions.  The  room  should  not  be  opened  up 
until  after  eight  hours  have  elapsed. 

Direct  sunlight  kills  tubercle  bacilli  even  when  they  are  enclosed 
in  sputum,  but  the  rapidity  with  which  they  are  killed  depends  some- 
what upon  the  season;  a  longer  exposure  is  required  in  winter  than 
in  summer.  Sputum  exposed  out  of  doors  in  indirect  light  may  remain 
infectious  for  some  time.  In  order  to  determine  that  tubercle  bacilli 
are  killed  it  is  necessary  to  inoculate  the  material  containing  them 
into  guinea-pigs,  the  guinea-pig  being  far  more  sensitive  than  artificial 
media  for  this  purpose.  Theobald  Smith2  has  shown  that  it  takes  at 
least  1500  times  as  many  tubercle  bacilli  to  infect  artificial  media 
as  it  does  to  infect  a  guinea-pig.  It  must  be  remembered  that  even 
killed  tubercle  bacilli,  as  Prudden  and  Hodenpyl3  have  shown,  produce 
tubercles  in  guinea-pigs,  but  that  these  tubercles  are  not  transmissible 

1  Theobald  Smith,  Jour.  Exp.  Med.,  1899,  iv,  233. 

2  Jour.  Med.  Research,  1913,  xxviii,  91. 

3  New  York  Med.  Jour.,  Jun  e6,  1891,  p.  20. 


TUBERCLE  BACILLUS  435 

to  other  guinea-pigs;  consequently  it  is  necessary  to  inoculate  a 
second  set  of  guinea-pigs  from  the  tubercles  developing  in  the  first 
set  of  pigs  in  order  to  be  certain  that  the  bacilli  are  killed. 

Products  of  Growth. — Enzymes. — Tubercle  bacilli  do  not  produce 
soluble  proteolytic  enzymes.  No  carbohydrate-splitting  enzymes 
have  been  observed. 

Carriere,1  Wells  and  Corper2  have  shown  that  the  bodies  of  tubercle 
bacilli  contain  a  lipase  of  moderate  activity.  Kendall  Walker  and 
Day3  have  demonstrated  that  the  filtrates  of  cultures  of  human  and 
bovine  tubercle  bacilli  contain  a  soluble  esterase;  the  action  of  the 
enzyme  upon  fats  is  relatively  slight.  This  esterase  is  produced  in 
an  active  form  in  media  of  very  simple  composition.4 

Winternitz  and  Meloy5  have  shown  that  the  lipase  (esterase  ?) 
activity  of  the  blood  is  decreased  in  tuberculosis.  Bauer  states  it  is 
increased  in  the  early  stages  of  the  disease. 

Hemolysis. — Raybaud  and  Hawthorn6  state  that  cultures  of  tubercle 
bacilli  will  not  hemolyze  the  erythrocytes  of  normal  guinea-pigs;  the 
erythrocytes  of  tuberculous  pigs  are  hemolyzed. 

Tubercle  bacilli  do  not  form  indol  or  the  ordinary  products  of  bac- 
terial decomposition  in  ordinary  media.  They  do  not  liquefy  gelatin 
nor  do  they  coagulate  milk.  Theobald  Smith7  has  called  attention 
to  a  very  constant  differential  character  between  the  human  and 
bovine  types  of  the  tubercle  bacillus.  In  glycerin  broth  the  human 
tubercle  bacillus  causes  a  permanent  acid  reaction,  while  the  bovine 
bacillus  under  the  same  conditions  causes  the  medium  to  become 
alkaline  if  the  growth  conditions  are  suitable.  Tuberculin  prepared 
from  human  cultures  consequently  is  acid  in  reaction,  while  that 
prepared  from  bovine  cultures  is  alkaline.  The  organism  liberates 
a  moderate  amount  of  ammonia  incidental  to  its  metabolism  of  pro- 
teins or  amino  acids.8  Old  cultures  of  tubercle  bacilli  occasionally 
are  very  gelatinous.9  Vaughan,10  White  and  Avery11  and  White,12  using 

1  Compt.  rend.  Soc.  Biol.,  1901,  liii,  320. 

2  Jour.  Inf.  Dis.,  1912,  xi,  388. 

3  Ibid.,  1914,  xv,  443. 

4  Kendall,  Walker  and  Day,  Jour.  Inf.  Dis.,  1914,  xv,  455. 

6  Jour.  Med.  Research,  1910,  xxii,  107. 

« Compt.  rend.  Soc.  Biol.,  1903,  No.  55. 

7  Trans.  Am.  Phys.,  1903,  xviii,  108;   Am.  Jour.  Med.  Sc.,  1904,  cxxviii,  216;   Jour. 
Med.  Research,  1905,  xiii,  253,  405. 

8  Kendall,  Day  and  Walker,  Jour.  Inf.  Dis.,  1914,  xv,  417,  423,  428,  433. 

9  Weleminsky,  Berl.  klin.  Wchnschr.,  1912,  xlix,  1320.     Gotzl,  Wien.  klin.  Wchnschr. 
1913,  1614.     Kendall,  Day  and  Walker,  Jour.  Inf.  Dis.,  1914,  xv,  428. 

10  Protein  Split  Products,  1913. 

11  Jour.  Med.  Research,  1912,  xxvi,  317. 

12  Trans.  9th  Ann.  Meet.  Nat'l.  Assn.  Study  and  Proven.  Tuberculosis. 


436  THE   TUBERCLE  BACILLUS  GROUP 

the  method  of  Vaughan,  have  isolated  a  non-specific  poisonous  sub- 
stance from  fat-free  tubercle  bacilli  which  kills  guinea-pigs  with 
symptoms  typical  of  anaphylaxis.  The  mineral  constituents  of 
tubercle  bacilli  have  been  determined  by  de  Schweinitz  and  Dorset.1 

Toxins.— The  tubercle  bacillus  appears  to  elaborate  both  an  endo- 
toxin  and  an  extracellular  toxin.2  The  endotoxin  causes  necrosis, 
caseous  degeneration  and  general  cachexia  and  stimulates  tubercle 
formation.  The  extracellular  toxin  causes  fever  and  the  acute  inflam- 
matory reaction  observed  around  tubercles  and  tuberculous  tissue 
in  tuberculous  animals.  Little  or  no  effect  is  produced  in  healthy 
animals  except  emaciation.  The  toxins  liberated  by  the  tubercle 
bacillus  are  apparently  on  the  whole  rather  mild,  because  they  produce 
as  a  rule  only  local  lesions.  This  would  indicate  that  the  diffusion 
of  toxin  is  somewhat  limited.  Furthermore,  the  kidneys  do  not  ordi- 
narily exhibit  anatomical  changes  which  could  be  definitely  ascribed 
to  the  elimination  of  a  tuberculous  toxin  through  them.  Whether 
the  cachexia,  which  is  a  prominent  feature  of  advanced  cases  of 
tuberculosis,  is  to  be  regarded  as  a  purely  toxic  phenomenon  is  not 
clear.  Holmes3  has  suggested  that  the  fatty  acids  of  the  tubercle 
bacillus  cause  a  lymphocytosis. 

Pathogenesis. —  Human. — According  to  Naegeli,4  rather  more  than 
90  per  cent,  of  adults  who  come  to  autopsy  show  scar  tissue  at  the 
apices  of  the  lungs,  which  he  believed  were  healed  tubercles.  Later 
observations  have  not  fully  confirmed  these  figures,  but  it  appears 
that  fully  50  per  cent,  of  adults  have  healed  tubercles  at  this  site.5 
Frequently  virulent  tubercle  bacilli  have  been  isolated  from  the 
centre  of  this  scar  tissue,  but  it  should  be  remembered  that  occasion- 
ally virulent  tubercle  bacilli  have  been  isolated  from  bronchial  lymph 
nodes  which  appear  to  be  normal. 

Modes  of  Infection. — Hereditary  Transmission. — Transmission  of 
the  tubercle  bacillus  through  the  sperm  has  never  been  established; 
transmission  through  the  ovum  is  also  not  definitely  established. 
The  maternal  blood,  on  the  contrary,  appears  to  be  a  vehicle  through 
which  tubercle  bacilli  may  pass,  or  grow  through  the  placental  barrier 
and  thus  reach  and  infect  the  fetus.6 

1  Jour.  Am.  Chem.  Assn.,  1898,  xx,  618. 

2  Armand-Delille,  Monographies  Cliniques  en  Medicine,  etc.,  1911,  No.  06,  Paris. 

3  Guy's  Hospital  Reports,  1909,  lix,  155. 
*  Virchow's  Arch.,  1900,  clx,  426. 

6  Lubarsch,  Virchow's  Arch.,  1913,  ccxiii,  417. 

6  See  Gartner,  Ztschr.  f.  Hyg.,  1893,  xiii,  126-139,  for  summary  and  discussion  of 
early  literature.  Also,  Schmorl  and  Geifel,  Miinchen.  med.  Wchnschr.,  1904,  1676. 


TUBERCLE  BACILLUS  437 

Latency  Theory. — Baumgarten1  believes  that  tubercle  bacilli  may 
lie  dormant  in  the  body  for  months  or  years  and  become  active  when 
the  u  resistance"  of  the  body  is  lowered.  The  evidence  is  on  the  whole 
opposed  to  this  view,  partly  because  congenital  tuberculosis  is  uncom- 
mon, chiefly  because  the  organs  of  fetuses  of  tuberculous  mothers 
do  not  cause  infection  in  guinea-pigs.  More  recently  v.  Behring2  has 
advanced  the  theory  that  infection  takes  place  in  childhood,  probably 
by  ingestion  of  milk  containing  tubercle  bacilli,  and  that  the  manifes- 
tations of  infection  become  apparent  later  in  life. 

Inoculation  Theory. — Direct  inoculation  through  the  skin  is  rare. 
Inhalation   Theory. — Droplet  infection  and  infection  by  dust  con- 
taining viable  tubercle  bacilli  appear  to  be  the  most  common  methods 
of  transmission  of  the  organism. 

Ingestion  of  milk  or  meat  containing  tubercle  bacilli  must  be  con- 
sidered as  important  methods  of  transmission  of  the  organism. 

Trauma,  establishing  a  locus  minoris  resistentiae  to  which  tubercle 
bacilli  lying  dormant  in  the  body  may  be  transported  and  set  up  infec- 
tion, is  probably  uncommon. 

Conditions  Favoring  Infection. — Overcrowding  with  its  attendant 
evils  of  dark,  damp  rooms,  poor  food  and  general  unhygienic  condi- 
tions appears  to  be  a  most  potent  factor  in  the  spread  of  tuberculosis. 
No  age  is  exempt,  although  the  disease  is  somewhat  less  frequent 
between  the  ages  of  five  and  ten  years,  greatest  between  sixteen  and 
thirty-five  years.  The  sexes  are  about  equally  infected.  Negroes 
are  especially  prone  to  the  disease,  possibly  because  of  their  surround- 
ings and  manner  of  living  rather  than  any  inherent  lack  of  resistance. 
Tuberculosis  is  relatively  uncommon  among  the  aboriginal  negro 
races  in  Africa.  Jews  appear  to  be  relatively  immune  to  the  disease. 
Those  occupations  in  which  dust  is  generated  in  large  amounts  exhibit 
a  higher  incidence  of  the  disease  than  occupations  in  which  dust  is 
not  a  feature.  Catarrhal  infections  of  the  respiratory  tract  appear 
to  predispose  to  pulmonary  tuberculosis  as  do  measles,  whooping- 
cough  and  influenza. 

Atria  of  Invasion. — The  respiratory  and  digestive  tracts  (including 
the  tonsils)  and  the  skin  are  the  three  portals  through  which  tubercle 
bacilli  enter  the  tissues  of  the  body.  Of  these  the  respiratory  tract 
is  more  frequently  involved.  Droplet  infection  is  by  far  the  most 
common  method  of  transmission  of  tubercle  bacilli;  dust-borne  infec- 
tion is  probably  relatively  uncommon. 

1  Deutsch.  med.  Wchnschr.,  1882,  No.  22. 

2  Ibid.,  1903,  692;  1904,  194. 


438 


THE  TUBERCLE  BACILLUS  GROUP 


Tubercle  bacilli  also  enter  the  intestinal  tract  and  they  may  pass 
through  the  intestinal  mucosa  without  leaving  any  trace  of  their 
passage,  particularly  if  they  be  suspended  in  fatty  menstrua,  as  butter 
or  cream.1  The  bovine  type  of  the  tubercle  bacillus  may  enter  through 
the  tonsils,  or  the  digestive  tract  occasionally.  Rarely,  tubercle  bacilli 
enter  through  the  skin,  usually  causing  somewhat  localized  epidermal 
proliferations  containing  tubercle  bacilli  in  small  numbers,  which 
are  sometimes  called  butcher's  warts,  postmortem  warts  or  verruca 
necrogenica.  Usually  they  remain  localized. 

COMBINED  TABULATION,  CASES  REPORTED  AND  OWN  SERIES  OF 
CASES.     (PARK  AND  KRUMWIEDE.) 


Diagnosis. 

Adults  16  years 
and  over 

Children 

5  to  16  years. 

Children 
under  5  years. 

Human. 

Bovine. 

Human. 

Bovine. 

Human. 

Bovine. 

Pulmonary  tuberculosis     .... 
Tuberculous     adenitis,     axillary     or 
inguinal    
Tuberculous  adenitis,  cervical 
Abdominal  tuberculosis     .... 
Generalized  tuberculosis,  alimentary 
origin        . 
Generalized  tuberculosis    .... 
Generalized    tuberculosis,    including 
meninges,  alimentary  origin 
Generalized    tuberculosis,    including 
meninges                                       . 

568 

2 
22 
15 

6 

28 

4 

18 
11 
1 

2 

1? 

1 
3 

1 

1 
1 

1 

11 

4 
33 

7 

2 
4 

1 

7 
2 
26 
1 
1 

20 

7 

3 
1 

1 
1 

12 

2 
15 
6 

13 

28 

3 

45 
14 
21 

1 
1 

20 
13 

10 
5 

8 

1 

2 

i 

Tubercular  meningitis        .... 
Tuberculosis  of  bones  and  joints 
Genito-urinary  tuberculosis    . 
Tuberculosis  of  skin           .... 

Miscellaneous  cases: 
Tuberculosis  of  tonsils     . 
Tuberculosis  of  mouth  and  cervi- 
cal nodes 

Tuberculous  sinus  or  abscesses  . 
Sepsis,  latent  bacilli   .... 

Totals  

677 

9 

99 

33 

161 

59 

Mixed  or  double  infections,  4  cases. 
Total  cases,  1042. 

For  some  years  much  discussion  has  centred  upon  the  incidence  of 
bovine  tubercle  bacillus  infection  in  man.  Koch  was  inclined  to  the 
view  that  infection  with  this  organism  was  so  rare  as  to  be  practically 
negligible.  Later  he  modified  his  opinion.  Weber2  studied  628  cases 

1  Nicolas  and  Descos,  Jour.  Physiol.  et  Path,  gen.,  1902,  iv,  910;  Ravenel,  Jour.  Med. 
Research,  1903,  x,  460. 

2  Tuberkulose,  Arbeiten  a.  d.  kais.  Gesamte,  1910,  Heft  x. 


TUBERCLE  BACILLUS  439 

(284  children,  335  adults,  9 — age  unstated),  all  of  whom  had  drunk 
the  milk  of  cows  having  tuberculosis  of  the  udder,  or  had  consumed 
uncooked  products  made  from  the  milk.  Only  two  patients,  both 
very  young  children,  were  definitely  shown  to  be  infected  with  bovine 
tubercle  bacilli.  Both  had  enlarged  caseous  cervical  glands  from  which 
the  organism  was  isolated  and  identified.  Six  children  and  one  adult 
had  glandular  swelling  in  the  neck,  but  the  evidence  was  not  conclusive 
that  bovine  infection  had  taken  place.  The  general  conclusion  was 
that  there  was  relatively  little  danger  from  drinking  milk  containing 
viable  bovine  tubercle  bacilli. 

Much  more  convincing  are  the  studies  of  Park  and  Krumwiede.1 
The  accompanying  table  (see  preceding  page),  which  is  their  sum- 
mary of  their  own  extensive  investigation  and  a  recapitulation  of 
authentic  observations  of  others,  shows  very  definitely  that  infection 
with  bovine  bacilli  is  relatively  common  in  children  and  young  adults 
up  to  sixteen  years  of  age,  but  relatively  uncommon  in  adults. 

Bovine  bacilli  are  found  not  only  in  unpasteurized  market  milk,2 
but  also  in  the  glandular  organs  of  a  considerable  proportion  of  cattle 
and  swine.  The  muscles  are  usually  not  invaded.  No  meat  from 
tuberculous  animals  can  be  offered  for  sale  in  the  public  markets, 
however. 

Lipschutz3  has  reported  a  case  of  cutaneous  infection  by  the  avian 
tubercle  bacillus  in  man  which  resembled  leprosy  anatomically.  The 
diagnosis  was  arrived  at  only  after  an  exhaustive  study  of  the  organism. 
Infection  of  man  with  the  avian  tubercle  bacillus  is  uncommon. 

The  mechanism  of  infection  with  the  tubercle  bacillus  has  been  the 
subject  of  much  controversy.  It  is  apparent  that  the  acid-fastness 
of  the  organism  per  se  does  not  confer  pathogenic  properties  upon  the 
organism  because  other  non-pathogenic  acid-fast  bacteria  are  unable 
to  induce  progressive  disease  from  man  to  man  or  from  animal  to 
animal.  Acid-fastness,  however,  may  be  an  initial  factor  in  patho- 
genism,  an  opening  wedge  as  it  were,  for  it  appears  to  be  well  estab- 
lished that  acid-fast  bacteria  are  relatively  insoluble  in  body  fluids 
and  remain  unchanged  for  considerable  periods  of  time  when  they 
are  introduced  into  the  animal  body.  Theobald  Smith4  has  advanced 
a  tentative  hypothesis  which  explains  satisfactorily  many  of  the 

1  Trans.  6th  Ann.  Meet.  Nat'l.  Assn.  Study  and  Preven.  Tuberculosis* 

2  See  Kober,  Trans.  Am.  Phys.  for  literature  to  1903.     Hess,  Jour.  Am.  Med.  Assn., 
1909,  Hi,  No.  13,  1011.     Moore,  Jour.  Med.  Research,  1911,  xxiv,  517. 

3  Arch.  f.  Dermat.  u.  Syph.,  June,  1914,  cxx. 

4  Jour.  Am.  Med.  Assn.,  April  28,  1906. 


440  THE  TUBERCLE  BACILLUS  GROUP 

phenomena.  As  tubercle  bacilli  reach  the  body  (and  as  they  escape 
from  the  body)  they  are  surrounded  by  a  protective  envelope  which 
causes  the  organism  to  behave  somewhat  as  an  inert  foreign  body 
until  it  finally  settles  down  in  some  structure  where  it  can  grow. 
The  envelope  is  then  slowly  removed  or  modified  by  the  action  of 
normal  tissue  fluids  and  growth  commences.  In  this  connection  it  is 
interesting  to  note  that  young  tubercle  bacilli  are  frequently  non- 
acid-fast,1  and  that  the  tissues  usually  invaded  by  the  bacilli — lym- 
phoid  tissue  and  the  lungs — contain  active  lipase.2  If  this  supposition 
is  correct,  the  tubercle  bacillus  may  remain  latent  in  the  body  until 
the  fatty  capsule  is  removed  or  modified,  perhaps  by  a  fat-splitting 
enzyme  (lipase) ;  then  development  takes  place.  It  should  be  remarked 
parenthetically  that  polymorphonuclear  leukocytes  which  occasion- 
ally engulf  tubercle  bacilli  do  not  contain  lipase;3  these  leukocytes 
may  transport  the  organisms  to  lymphoid  tissue  or  other  tissue  where 
eventually  the  bacilli  escape,  thus  establishing  new  foci.  Mono- 
nuclear  leukocytes  appear  to  contain  lipase,  as  do  certain  fixed  phago- 
cytic  cells  in  the  alveoli  of  the  lungs. 

Lenk  and  Pollak4  and  Wiener5  appear  to  have  found  active  proteo- 
lytic  ferments  in  tuberculous  exudates.  Opie  and  Barker6  have  shown 
that  the  mononuclear  epithelioid  cells  contain  an  enzyme  which  digests 
protein  in  a  slightly  acid  medium;  it  is  practically  inert  in  an  alkaline 
medium.  Jobling  and  Petersen7  have  found  that  the  inhibition  of 
enzyme  action  in  caseous  tubercle  foci  is  apparently  due  to  unsaturated 
fatty  acids.  Saturation  of  these  acids  with  iodin  causes  an  accelera- 
tion of  the  activity  of  the  ferments. 

The  primary  lesions  usually  tend  to  progress  slowly.  Secondary 
invasion  by  tubercle  bacilli  through  the  lymph  and  bloodvessels 
frequently  occurs,  causing  tuberculous  foci  in  various  ducts  and 
glands  of  the  body,  as  the  bronchi,  alveoli  of  the  lungs,  spleen,  liver, 
tubules  of  the  kidney,  and  in  the  genito-urinary  system,  particularly 
the  epididymis  and  testicle  of  the  male  and  the  Fallopian  tubes  in  the 
female.  The  glandular  organs  are  those  most  commonly  infected, 
and  of  these  the  lungs  and  lymph  nodes  are  most  frequently  involved; 

1  Wolbach  and  Ernst,  Jour.  Med.  Research,  1903,  x,  313. 

2  Bradley,  Jour.  Biol.  Chem.,  1913,  xiii,  No.  4.     Briscoe,  Jour.  Path,  and  Bact.,  1907, 
xii.     Bartel  and  Neumann,  Centralbl.  f.  Bakt.,  Orig.,  1909,  xlviii,  657.     Zinsser  and 
Carey,  Jour.  Am.  Med.  Assn.,  1912,  Iviii,  692. 

3  Fiessinger  and  Marie,  Compt.  rend.  Soc.  Biol.,  1909,  Ixvii,  177.     Bergell,  Miinchen 
med.  Wchnschr.,  1909,  Ivi,  64. 

4  Deutsch.  Arch.  klin.  Med.,  1910,  cix,  350.  5  Biochem.  Ztschr.,  1912,  xli,  149. 
6  Jour.  Exp.  Med.,  1908,  x,  645;    1909,  xi,  686.       7  Ibid.,  1914,  xix,  383. 


TUBERCLE  BACILLUS  441 

also  the  spleen,  kidneys,  liver,  meninges  both  of  the  cord  and  brain, 
the  pleural  and  pericardial  cavities,  the  genito-urinary  apparatus, 
and,  less  frequently,  joints  and  bones.  The  muscles  are  only  very 
rarely  invaded.  Various  clinical  names  have  been  applied  to  tuber- 
culosis of  different  tissues:  tuberculosis  of  the  lungs  is  commonly 
designated  consumption;  of  the  spine,  Pott's  Disease;  of  the  cervical 
lymph  glands,  scrofula;  and  of  the  skin,  lupus.  The  characteristic 
initial  lesion  is  a  small  nodule  or  tubercle  which  may  undergo  secon- 
dary changes,  as  caseation,  calcification,  ulceration,  or  various  types 
of  sclerosis.  In  the  lungs  the  first  organisms  that  reach  the  alveoli 
may  leave  no  trace.  They  are  dissolved  there  apparently,  but  may 
produce  no  progressive  lesion.  A  second  invasion  in  the  same  area 
frequently  causes  a  local  inflammation  which  usually  results  in  infec- 
tion, apparently  because  the  body  has  been  sensitized  by  the  first 
bacilli  that  entered,  and  in  some  way  is  rendered  locally  susceptible 
to  the  organism. 

The  irritation  caused  by  the  extracellular  toxin  excreted  by  the 
tubercle  bacillus  brings  about  a  response  on  the  part  of  the  tissues 
which  is  protective,  as  is  manifested  by  a  walling  off  of  the  bacilli. 
First  there  is  a  proliferation  of  the  connective  tissue  which  forms  a 
spherical  mass  of  epithelioid  cells  around  the  focus  of  infection.  Out- 
side of  the  epithelioid  cells  there  is  usually  an  infiltration  of  lympho- 
cytes. The  tissue  is  avascular  and  the  young  tubercles  contain  little 
or  no  fats.1  The  central  part  of  the  tubercle  soon  begins  to  undergo 
coagulation  necrosis,  probably  due  to  the  action  of  the  intracellular 
toxin,  and  it  is  gradually  converted  into  a  homogeneous,  cheesy  mass. 
In  many  tubercles  giant  cells  are  found,  which  are  formed  either  by 
the  coalescence  of  several  epithelioid  cells,  or  by  atypical  cell  divi- 
sion, the  nucleus  dividing  faster  than  the  cytoplasm.  The  nuclei  of 
the  giant  cell  are  arranged  peripherally  as  a  rule,  either  completely 
around  the  cell,  or  in  the  shape  of  a  horseshoe.  The  centre  of  the 
giant  cell  likewise  may  undergo  caseous  degeneration,  and  tubercle 
bacilli  are  not  infrequently  found  in  the  middle  of  these  cells.2  Accord- 
ing to  Zeit,  giant  cells  are  essentially  blind  blood  capillaries  which 
have  extended  into  the  tuberculous  area,  but  have  not  become  true 
vessels  because  the  toxins  of  the  organisms  have  prevented  the 
final  development  of  functional  blood  channels.  Besides  these  small 

1  Joest,  Virchow's  Arch.,  1911,  cciii,  451. 

2  See  Evans,  Bowman,  and  Winternitz,  Jour.  Exp.  Med.,  1914,  xix,  283,  for  a  critical 
experimental  study  of  the  histogenesis  of  the  miliary  tubercle  in  vitally  stained  rabbits 
for  the  finer  details  of  the  process. 


442  fH%  TUBERCLE  BACILLUS 


miliary  tubercles,  larger  areas  of  caseation  may  develop;  epithelioid 
cells,  lymphocytes,  and  giant  cells  are  usually  found  closely  packed 
around  these  areas. 

The  destruction  of  the  capillaries  and  the  resulting  avascular  tissue 
helps  in  the  necrosis  of  the  tubercle  by  cutting  off  the  blood  supply. 

What  is  generally  known  as  consumption  or  destruction  of  the  lung 
tissue  is  probably  not  due  to  the  action  of  the  tubercle  bacillus  alone, 
but  to  secondary  infection  and  liquefaction  of  tissue  by  other  organ- 
isms, as  the  streptococcus,  staphylococcus,  pneumococcus,  or  Micro- 
coccus  tetragenus.  If  a  caseous  necrotic  tubercle  located  near  a 
bronchus  ruptures  into  this  bronchus,  a  large  amount  of  tuberculous 
material  is  suddenly  swept  into  the  regional  areas  of  the  lung,  over- 
whelming it  and  setting  up  a  rapidly  fatal  infection  which  is  known 
as  galloping  consumption  or  phthisis  florida.  If  a  caseous  tubercle 
ruptures  into  a  lymph  or  bloodvessel,  the  material  may  be  carried 
very  widely  through  the  body,  causing  generalized  miliary  tuber- 
culosis, which  resembles  typhoid  fever  clinically.  Hemorrhage  not 
infrequently  takes  place  from  the  lung,  due  to  the  erosion  and  subse- 
quent bursting  of  a  bloodvessel  which  may  have  been  included  in  the 
caseous  area.  In  the  human  lung  it  is  practically  always  possible  to 
find  old  lesions  at  the  apices  when  the  infection  is  due  to  the  human 
type  of  the  tubercle  bacillus.  Uncommonly  no  old  healed  tubercles 
can  be  found,  and  the  lungs  are  filled  with  miliary  tubercles,  in  which 
case  the  infection  is  usually  caused  by  the  bovine  type  of  the  tubercle 
bacillus.  Tubercle  bacilli  ingested  with  milk  or  other  foods  may  cause 
tubercle  formation  in  the  mesenteric  glands  with  lesions  in  other  parts 
of  the  body.  Metastatic  nodules  are  found  occasionally  in  the  brain, 
meninges,  and  epiphyses  of  bones. 

Pathogenesis  for  Lower  Animals.  —  Generally  speaking,  the  human 
type  of  the  tubercle  bacillus  is  less  virulent  for  lower  animals  than  the 
bovine  type.  Monkeys  in  captivity,  however,  are  susceptible  to 
both  types,  and  even  infection  with  the  avian  type  has  been  found 
in  them.  The  course  of  the  disease,  which  is  spontaneous,  runs  similar 
to  that  of  human  consumption,  with,  however,  a  greater  tendency 
toward  generalized  invasion.  Goats,  sheep,  and  horses  are  not  as  a 
rule  infected  with  the  human  tubercle  bacillus.  Cattle  are  very 
rarely  infected  with  the  human  type.  Dogs  and  cats  are  said  to  be 
infected  occasionally. 

Rabbits.  —  Rabbits  are  not  as  susceptible  to  the  human  tubercle 
bacillus  as  the  guinea-pig.  Subcutaneous  injections  of  the  human 


TUBERCLE  BACILLUS  443 

» 

tubercle  bacillus  usually  causes  only  local  lesions,  which  in  the  vast 
majority  of  instances  are  not  fatal  and  clear  up  after  some  weeks. 
Massive  doses,  however,  usually  produce  lesions.  Intravenous  injec- 
tion, unless  massive  doses  are  given,  also  fails  to  kill  rabbits  as  a  rule. 
Occasionally,  however,  a  generalized  tuberculosis  with  fatal  termina- 
tion results.  Intraperitoneal  inoculations  only  occasionally  bring 
about  a  generalized  fatal  tuberculosis.  Usually  slight  lesions  are  pro- 
duced which  clear  up  spontaneously.  Ingestion  of  human  organisms 
rarely  leads  to  infection.1 

Guinea-pigs. — Guinea-pigs  are  very  susceptible  to  infection  with 
either  the  human  or  bovine  tubercle  bacilli,  although  the  disease 
rarely  appears  spontaneously.  Theobald  Smith2  has  shown  that  it 
requires  but  one  fifteen-hundredth  as  much  tuberculous  material  to 
infect  a  guinea-pig  as  is  required  to  infect  artificial  media.  This 
susceptibility  of  the  guinea-pig  to  inoculation  with  the  tubercle  bacil- 
lus explains  the  well-attested  fact  that  inoculation  of  suspected  tuber- 
culous material  into  these  animals  is  a  far  more  delicate  test  for  their 
presence  than  attempts  to  grow  the  organisms  from  the  same  material 
on  artificial  media.  It  must  be  remembered  in  this  connection  that 
even  dead  tubercle  bacilli  stimulate  tubercle  formation  in  guinea-pigs,3 
hence  for  an  absolutely  safe  diagnosis  whatever  tubercles  are  pro- 
duced in  the  first  guinea-pig  must  be  ground  up  and  injected  into  a 
second  guinea-pig.  If  viable  tubercle  bacilli  are  present  a  successful 
infection  will  take  place,  otherwise  the  experiment  is  negative. 

Subcutaneous  Inoculation. — After  ten  to  fourteen  days  a  small  hard 
nodule  appears  at  the  site  of  inoculation,  and  very  soon  afterward  the 
regional  lymph  glands  begin  to  enlarge  and  the  animal  begins  to  lose 
weight.  The  animal  usually  dies  in  from  two  to  four  months.  Post- 
mortem, the  spleen  is  enlarged,  yellowish-brown  in  color,  and  studded 
with  tubercles,  some  minute  and  gray,  others  larger,  yellowish  and 
frequently  caseous.  The  regional  lymph  nodes  also  have  usually 
undergone  caseation,  particularly  the  inguinal  glands,  less  com- 
monly the  axillary  glands.  The  liver  usually  has  a  few  rather  large 
caseous  or  fibrinous  tubercles,  particularly  on  the  free  border.  The 
kidneys  also  may  have  a  few  tubercles.  If  the  animal  has  lived  two 
or  three  months  the  thoracic  cavity  is  also  invaded,  and  scattered 
miliary  tubercles  may  be  seen  on  the  lungs.  The  mesenteric,  bronchial, 
sternal  and  cervical  glands  are  invaded. 

1  Theobald  Smith,  Jour.  Med.  Research,  1905,  xiii,  253. 

2  Jour.  Med.  Research,  1913,  xxviii,  91. 

1  Prudden  and  Hodenpyl,  New  York  Med.  Jour.,  1901,  and  others. 


444  THE  TUBERCLE  BACILLUS  GROUP 

Intraperitoneal  Inoculation. — The  disease  runs  a  more  rapid  course, 
death  usually  taking  place  in  from  three  to  eight  weeks.  The  peri- 
toneum is  chiefly  involved,  particularly  when  death  takes  place  early. 
The  omentum  is  thickly  studded  with  tubercles  which  tend  to  become 
confluent  and  to  caseate.  Certain  mesenteric  glands  also  enlarge  and 
become  caseous.  As  in  the  subcutaneous  inoculation,  the  inguinal 
and  axillary  glands  may  be  involved,  but  the  lesions  do  not  progress 
so  far. 

Ingestion. — The  lesions  usually  resemble  those  of  intraperitoneal 
infection,  with,  as  a  rule,  more  marked  lung  involvement. 

Inhalation  and  Pulmonary  Inoculation. — The  lungs  contain  con- 
fluent tubercles,  many  of  which  are  caseated.  Not  infrequently  one 
or  more  entire  lobes  may  be  involved.  Cavity  formation,  however, 
is  uncommon.  The  abdominal  viscera,  particularly  the  spleen,  are 
involved,  as  well  as  the  regional  lymph  glands. 

Products  of  Clinical  Importance  Derived  from  the  Tubercle  Bacillus. 
— Old  Tuberculin  (O.  T.  Koch). — Four  to  six  weeks'  pure  culture  of  the 
tubercle  bacillus  grown  in  5  per  cent,  glycerin  broth  is  killed  by  heat- 
ing to  110°  C.  for  half  an  hour,  and  then  evaporated  to  one-tenth  its 
original  volume  on  the  steam  bath.  It  is  then  filtered  through  sterile, 
unglazed  porcelain  filters.  The  resulting  fluid,  which  is  dark  brown 
in  color,  syrupy  in  consistency,  and  which  keeps  in  the  undiluted  con- 
dition in  the  cold  and  away  from  sunlight  for  months  apparently 
unchanged,  is  known  as  old  tuberculin.  Old  tuberculin  contains  the 
water  and  glycerin-soluble  products  of  metabolism  of  the  tubercle 
bacillus  and  products  of  autolysis  of  tubercle  bacilli  which  are  not 
precipitated  by  heat,  as  well  as  unchanged  concentrated  constituents 
of  the  broth  and  about  50  per  cent,  of  glycerin:  0.25  to  0.50  per 
cent,  tricresol  is  added  as  a  preservative.  The  nature  of  the  reactive 
substance  or  substances  in  tuberculin  is  unknown.  The  composition 
of  tuberculin  even  when  prepared  by  a  uniform  technic  appears  to 
be  variable.1  Tuberculin  prepared  from  the  human  type  of  the  tuber- 
cle bacillus  is  acid  in  reaction;  that  from  the  bovine  type  is  alkaline.2 

THE  NATURE  OF  TUBERCULIN. — Tuberculin3  appears  to  be  a  true 
product  of  the  metabolism  of  the  tubercle  bacillus.  It  is  thermostabile, 
dialyzable,  insoluble  in  alcohol,  gives  no  biuret  reaction,  and  is  pre- 

1  White  and  Hollander,  The  Chemical  Composition  of  Commercial  Tuberculins,  Trans. 
9th  Ann.  Meet.  Natl.  Assn.  Study  and  Preven.  Tuberculosis. 

2  Theobald  Smith,  Jour.  Med.  Research,  1905,  xiii,  405. 

3  The  word  "tuberculin"  appears  to  have  been  used  first  by  Pohl-Pincus,  Deutsch. 
med.  Wchnschr.,  1884,  108. 


TUBERCLE  BACILLUS  445 

cipitated  by  certain  alkaloidal  precipitants  as  tannic  acid,  potassium 
mercuric  iodide  and  mercuric  chloride  in  acid  solution.  It  is  decom- 
posed by  pepsin  HC1  and  by  trypsin  in  alkaline  media.  Proskauer 
and  Beck,1  and  Lowenstein  and  Pick,2  and  others  have  shown  that 
tuberculin  is  produced  by  the  tubercle  bacillus  when  this  organism  is 
grown  in  a  protein-free  medium.  They  suggest  that  it  is  probably  a 
polypeptid. 

Whether  tuberculin  contains  a  true  toxin  or  an  endotoxin,  or  a 
mixture  of  both  toxin  and  endotoxin  is  not  clearly  settled.  Pick 
believes  it  contains  a  true  toxin  secreted  by  the  tubercle  bacillus. 

Variants  of  Old  Tuberculin. — A  number  of  observers,  impressed  with 
the  possibility  that  the  reactive  substances  of  tuberculin  might  be 
changed  by  heat,  have  attempted  to  produce  tuberculin  which  has 
been  unheated. 

(a)  Bouillon  Filtrate    Denys   (B.   F.). — This  is  unheated,  uncon- 
centrated  old  tuberculin  prepared  as  above  and  sterilized  by  passage 
through  sterile  porcelain  filters. 

(b)  Vacuum  Tuberculin. — The  six  weeks  glycerin  broth  culture  of 
the  tubercle  bacillus  is  concentrated  to  one-tenth  its  volume  in  vacuo 
and   filtered.     By   so   doing   the   advantages   of   concentration    are 
obtained  without  the  disadvantages  of  heating. 

The  action  of  old  tuberculin  and  its  variants  would  suggest  that  it 
does  not  contain  all  the  necessary  elements  for  the  establishment  of 
true  immunity,  and  this  has  led  to  the  production  of  a  series  of  new 
products,  new  tuberculins,  which  attempt  to  retain  the  more  specific 
products  of  the  tubercle  bacillus.  The  principle  involved  is  to  grind 
up  dried  tubercle  bacilli  in  ball  mills  to  an  impalpable  powder,  and  to 
suspend  or  partially  dissolve  this  powder  in  salt  solution  with  or 
without  the  addition  of  glycerin. 

New  Tuberculin  (T.  R.  Koch). — Young  virulent  tubercle  bacilli  are 
dried  first  between  sheets  of  sterile  filter  paper,  then  in  vacuo  over 
sulphuric  acid  and  phosphorus  pentoxide  until  thoroughly  anhydrous, 
then  ground  in  a  mortar  until  a  dry  powder  is  obtained.  This  powder 
is  suspended  in  water,  thoroughly  mixed,  and  then  centrifugalized. 
The  first  supernatant  fluid  obtained  (T.  O.)  is  rejected.  This  pre- 
liminary grinding  with  water  is  intended  to  wash  out  the  water-soluble 
substance.  The  residue  is  then  repeatedly  ground  and  centrifugalized, 
saving  the  supernatant  liquid  each  time  until  all  of  it  has  passed  into 

1  Ztschr.  f.  Hyg.,  1894,  xviii,  128. 
2Biochem.  Ztschr.,  1911,  xiii,  142. 


446  THE   TUBERCLE  BACILLUS  GROUP 

solution  and  suspension.  This  constitutes  new  tuberculin.  The  new 
tuberculin  is  finally  prepared  of  such  a  strength  that  1  c.c.  of  the 
dried  residue  will  contain  0.002  gram  of  solid  material.  It  is  cus- 
tomary to  add  glycerin  and  a  small  amount  of  formaldehyde  to  the 
preparation  before  it  is  finally  made  up  to  strength. 

At  times  new  tuberculin  has  been  found  to  contain  living,  virulent 
tubercle  bacilli,  although  they  are  killed  by  prolonged  exposure  to 
formalin.  New  tuberculin  was  originally  intended  for  curative  pur- 
poses, as  it  was  found  to  be  relatively  free  from  the  toxic  sub- 
stances which  are  found  in  old  tuberculin.  The  theoretical  inherent 
dangers  of  this  preparation,  however,  have  tended  in  the  past  to  limit 
its  use. 

Bacillus  Emulsion  (B.  E.). — This  is  an  emulsion  of  untreated  tubercle 
bacilli  which  are  washed  and  dried  as  for  new  tuberculin.  The  organ- 
isms are  ground  thoroughly  and  then  suspended  by  continual  grinding 
in  physiological  salt  solution  containing  about  20  per  cent,  of  glycerin. 
From  0.25  to  0.5  per  cent,  carbolic  acid  is  added  to  kill  whatever 
tubercle  bacilli  or  other  organisms  may  have  been  included  in  the 
preparation.  For  use  it  is  standardized  so  that  1  c.c.  of  the  solution 
contains  the  equivalent  of  0.001  gram  of  dried  tubercle  bacilli.  It 
will  be  seen  that  the  bacillus  emulsion  contains  both  new  tuber- 
culin and  a  certain  amount  of  the  water-soluble  products  of  the 
tubercle  bacillus  or  old  tuberculin. 

Alkaline  Tuberculin  (T.  A.). — Virulent  tubercle  bacilli  freed  from 
culture  media  are  extracted  with  10  per  cent,  caustic  soda  for  three  to 
four  days  at  20°  C.  The  bacilli  and  their  fragments  are  then  removed 
by  filtration  through  filter  paper.  The  filtrate  is  neutralized  by  the 
careful  addition  of  hydrochloric  acid  and  again  filtered.  The  clear 
fluid  (T.  O.)  gives  similar  but  somewhat  more  severe  reactions  than 
the  regular  tuberculin.  It  often  leads  to  sterile  abscess  formation.1 

DIAGNOSIS   OF   TUBERCULOSIS. 

A.  Clinical,  by  tuberculin  reaction. 

1.  Action  of  tuberculin  on  healthy  animals  and  man. 

2.  Action  of  tuberculin  on  tuberculous  animals  and  man. 

3.  Principle  of  tuberculin  reaction. 

1  For  a  general  survey  of  the  nature  and  composition  of  various  tuberculins  see  Kuthy 
and  Wolff-Eisner,  Die  Prognosenstellung  bei  der  Lungentuberkulose,  Berlin  and  Vienna. 
1914,  pp.  438-446.  " 


DIAGNOSIS  OF  TUBERCULOSIS  447 

4.  Technic  of  tuberculin  reaction. 

(a)  Subcutaneous  test  (Koch). 

(b)  Cutaneous  test  (von  Pirquet). 

(c)  Percutaneous  test  (Moro). 

(d)  Detre  test  (human  and  bovine  tuberculin  to  detect 

the  type  of  infection). 

(e)  Ophthalmo  reaction  (Calmette  and  Wolff-Eisner) . 

5.  Specificity  of  the  tuberculin  reaction. 

B.  Serological. 

1.  Opsonic  index. 

2.  Agglutination. 

3.  Complement  fixation. 

C.  Bacteriological. 

1.  Principle  involved. 

(a)  Microscopical. 

(6)  Cultural. 

(c)  Animal  inoculation. 

2.  Technic. 

A.  Clinical  Diagnosis. — 1.  Action  of  Tuberculin  on  Healthy  Animals 
and  Man. — In  healthy  laboratory  animals,  as  guinea-pigs  and  rabbits, 
as  much  as  1  c.c.  of  old  tuberculin  may  be  injected  with  no  apparent 
harm  other  than  a  somewhat  transient  rise  in  temperature.  In  normal 
man  even  as  small  an  amount  as  0.01  c.c.  of  old  tuberculin  may  cause 
violent  symptoms:  chill,  temperature,  vomiting,  malaise,  and  even 
diarrhea.  These  effects,  however,  are  usually  transient.  Man  appears 
to  be  far  more  sensitive  to  tuberculin  than  the  guinea-pig. 

2.  Action  of  Tuberculin  on  Tuberculous  Animals  and  Man. — In  tuber- 
culous animals  very  small  amounts  of  tuberculin  injected  subcutan- 
eously  may  cause  marked   symptoms;   0.2   to   0.5  c.c.  will  almost 
invariably  kill  a  guinea-pig  which    has  been  injected  with  tubercle 
bacilli  from  four  to  five  weeks  before    by  the  subcutaneous  route. 
Intracerebral  inoculation  of  tuberculin  will  kill  tuberculous  guinea- 
pigs  in  much  smaller  amounts.    Postmortem  there  is  intense  conges- 
tion around  the  tuberculous  foci  and  ecchymotic  hemorrhages  in  the 
viscera.     Twenty-five   hundredths   (0.25)  of  a  cubic  centimeter  of 
old  tuberculin  would  be  extremely  dangerous  to  inject  into  a  tuber- 
culous man.    It  would  probably  result  fatally.1 

3.  The  Principle  Involved. — The  reaction  obtained  in  tuberculous 
man  or  animals  by  the  injection  of  tuberculin  or  other  products  of 

1  Deist,  Beitr.  z.  Klinik  d.  Tuberkulose,  1912,  xxii,  547,  has  observed  albumoses  in  the 
urine  of  tuberculous  patients  following  the  injection  of  tuberculin. 


448  THE  TUBERCLE  BACILLUS  GROUP 

the  tubercle  bacillus  depends  upon  the  fact  that  the  tuberculous 
subject  is  sensitized  to  the  proteins  of  the  tubercle  bacillus  as  the 
result  of  infection  with  this  organism.1  The  presence  of  the  proteins 
of  the  tubercle  bacillus  and  perhaps  other  products  of  growth  of  the 
tubercle  bacillus  stimulate  certain  body  cells  of  the  host  to  produce 
specific  proteolytic  ferments  which  dissolve  these  proteins  within  the 
body.2  When  tuberculin  is  introduced  into  the  tuberculous  host  these 
specific  proteolytic  ferments  liberate  from  the  tuberculin  a  poisonous 
cleavage  product  which  has  three  specific  effects:  a  focal  effect, 
characterized  by  intense  irritation  and  inflammation  around  the  tuber- 
culous foci,  a  local  effect,  and  a  general  effect  which  is  characterized 
by  a  rise  of  temperature  and  other  general  systemic  reactions.  The 
tuberculin  reaction  is  an  anaphylactic  reaction  according  to  Vaughan.3 

4.  Technic  of  the  Tuberculin  Reaction.4 — The  tuberculin  reaction 
elicited  in  man  by  the  introduction  of  tuberculin  is  of  two  types, 
depending  upon  the  method  of  injection  employed.  If  introduced 
subcutaneously  so  that  the  tuberculin  enters  the  lymph  or  blood 
streams,  even  in  minute  amounts,  the  reactions  consist  of  three  rather 
distinct  phases:  a  general,  a  local,  and  a  focal  reaction  respectively. 
If,  on  the  contrary,  the  injection  is  purely  superficial  in  the  epidermis 
or  on  the  conjunctiva  the  reaction  is  almost  exclusively  local. 

(a)  The  Subcutaneous  Reaction  ( Koch) . — The  characteristic  response 
following  the  subcutaneous  injection  of  appropriate  amounts  of  old 
tuberculin  is  a  generalized  reaction  consisting  of  a  rise  in  temperature 
of  at  least  one-half  a  degree  above  the  highest  temperature  exhibited 
before  the  inoculation.  This  rise  in  temperature  usually  begins  twelve 
to  eighteen  hours  after  the  injection;  it  may  be  delayed  to  twenty- 
four  or  even  forty-eight  hours.  The  temperature  should  be  taken  at 
half-hourly  intervals.  There  is  frequently  an  initial  chill  following 
the  introduction  of  tuberculin  and  in  addition  malaise,  headache  and 
restlessness;  even  nausea  or  vomiting  may  be  observed.  The  focal 
reaction  consists  essentially  of  hyperemia  and  a  distinct  inflammatory 
reaction  around  active  foci.  In  superficial  foci,  as  in  lupus,  this 
inflammatory  reaction  may  be  distinctly  seen,  and  in  deeper  foci 

1  For  discussion  of  theories  of  the  tuberculin  reaction,  see  Kuthy  and  Wolff-Eisner, 
Die  Prognosenstellung  bei  den  Lungentuberkulose,  Berlin  and  Vienna,  1914. 

2  White,  Jour.  Med.  Research,  1914,  xxx,  393,  has  shown  that  lipoids  of  the  tubercle 
bacillus  neither  sensitize  nor  induce  anaphylaxis  in  experimental  animals. 

3  Protein  Split  Products,  Philadelphia,  1913.     See  Baldwin,  Yale  Med.  Jour.,  February, 
1909,  for  excellent  summary  of  present  status  of  subject. 

4  The  diagnostic  and  particularly  the  therapeutic  use  of  tuberculin  requires  much 
skill  and  experience.     For  details,  see  Baldwin  and  Brown,  Osier's  Modern  Medicine, 
iii,   137,  361. 


DIAGNOSIS  OF  TUBERCULOSIS  449 

it  can  be  frequently  demonstrated  or  at  least  inferred  by  an  increase 
of  local  clinical  signs.  The  local  reaction  at  the  site  of  inoculation 
consists  essentially  of  a  reddened,  swollen,  circumscribed  area  of 
inflammation.  The  specificity  of  the  reaction  is  dependent  upon  the 
size  of  the  dose  of  tuberculin.  Too  large  a  dose  may  cause  a  reaction 
even  in  a  non-tuberculous  subject.  It  is  obvious  that  patients  already 
exhibiting  a  febrile  reaction  due  to  intercurrent  disease  or  otherwise 
are  unfit  subjects  for  injection,  for  the  rise  in  temperature  is  the  chief 
diagnostic  symptom  relied  upon  in  establishing  a  diagnosis. 

The  subcutaneous  injection  of  old  tuberculin  is  used  only  for  adults 
and  for  children  over  five  years  of  age  in  the  very  early  stages  of  the 
disease.  This  reaction  is  claimed  by  some  observers  to  be  more  deli- 
cate than  any  other  tuberculin  test.  In  practice  tuberculin  is  intro- 
duced subcutaneously  either  in  the  breast  or  preferably  in  the  back, 
and  a  control  inoculation,  using  dilute  glycerin  containing  0.5  per 
cent,  carbolic  acid,  is  made  in  another  area.  If  a  nodule  and  con- 
gestion appear  at  the  site  of  inoculation  of  the  tuberculin  and  the 
control  area  remains  practically  unchanged,  the  reaction  is  considered 
positive  if  the  temperature  chart  taken  at  half-hour  intervals  shows 
at  least  half  a  degree  rise  in  temperature  above  that  exhibited  pre- 
viously for  several  days.  The  size  of  the  dose  to  be  administered 
depends  upon  the  age  and  condition  of  the  patient  and  upon  the 
potency  of  the  old  tuberculin. 

(6)  The  Cutaneous  Test  (von  Pirquet). — The  patient's  forearm  is 
sterilized  and  two  drops  of  undiluted  old  tuberculin  are  placed  upon 
the  skin  about  8  to  10  c.m  apart.  A  light  scarification  is  made,  pre- 
ferably with  the  von  Pirquet  scarifier,  through  each  drop  of  tuberculin. 
A  control  scarification  is  made  midway  between  the  drops,  but  no 
tuberculin  is  applied  here.  A  small  pledget  of  cotton  is  placed  over 
each  drop  of  tuberculin  and  allowed  to  remain  ten  minutes  to  prevent 
the  tuberculin  from  spreading  beyond  the  site  of  scarification.  The 
amount  of  cotton  used  should  be  small  enough  to  prevent  any  con- 
siderable absorption  of  the  tuberculin.  No  dressing  is  required.  Dur- 
ing the  first  few  hours  of  vaccination  the  control  and  vaccinated  areas 
appear  the  same,  a  slight  area  of  inflammation  due  to  trauma  sur- 
rounding each.  The  specific  reaction  appears  first  as  a  slightly 
elevated  red  area  around  each  drop  of  tuberculin,  which  increases  in 
size  and  somewhat  in  elevation  until  it  reaches  a  diameter  of  from 
1  to  3  or  even  4  cm.,  the  former  being  the  more  common.  The 
maximum  intensity  is  usually  reached  within  forty-eight  hours,  the 

29 


450  THE   TUBERCLE  BACILLUS  GROUP 

first  signs  appearing  in  from  four  to  twenty-four  hours.  In  scrofulous 
children  small  raised  follicular  swellings  commonly  appear  around  the 
central  specific  area,  the  so-called  scrofulous  reaction.  It  should  be 
remembered  that  a  second  injection  of  tuberculin  in  the  same  area  on 
the  same  arm  following  an  initial  negative  reaction  may  be  positive. 
This  is  not  an  indication  of  infection,  however;  it  is  rather  a  mani- 
festation of  local  sensitization.1 

(c)  The  Percutaneous   Test  (Moro). — Moro  has  modified   the  von 
Pirquet  test  in  such  a  manner  as  to  exclude  the  traumatism  incidental 
to  scarification.    This  is  accomplished  by  rubbing  into  the  skin  of  the 
abdomen  or  the  chest  an  ointment  made  of  5  c.c.  of  old  tuberculin 
mixed  intimately  in  5  grams  of  anhydrous  lanolin.    In  practice  a  bit 
of  the  ointment  one-half  a  centimeter  in  diameter  is  rubbed  over  an 
area  of  about  four  square  inches  for  about  half  a  minute.    The  oint- 
ment is  left  on  the  skin  to  absorb  gradually.     A  positive  reaction 
consists  in  the  development,  usually  within  twenty-four  to  forty-eight 
hours,  of  a  number  of  small  red  papules  within  the  area  of  inunction. 
Ordinarily  but  a  few  papules  are  formed;  less  commonly  a  consider- 
able crop  appear.    Rarely  the  skin  in  the  immediate  area  is  reddened 
and  there  may  be  slight  itching.    The  papules  are  few  in  number  in 
a  mild  reaction,  usually  from  two  to  eight  or  ten;  they  are  red  and  from 
1  to  2.5  mm.  in  diameter.    A  moderate  reaction  is  characterized  by 
many  red  papules,  from  10  to  100,  which  are  rather  closely  crowded 
together  and  usually  from  0.25  to  2.5  mm.  in  diameter.    The  inter- 
papular  areas  of  skin  may  or  may  not  be  reddened.     If  the  skin  is 
reddened  it  frequently  itches  somewhat.    In  a  severe  reaction  papules 
appear  within  a  few  hours  after  inunction,  many  in  number  with  a 
markedly   hyperemic   background.      Itching   is    a  very  disagreeable 
feature  of  a  severe  reaction. 

(d)  The   Detre   Test. — Human  tuberculin  is  rubbed  into  one  arm 
and  bovine  tuberculin -is  rubbed  into  the  other,  using  preferably  the 
cutaneous  reaction  of  von  Pirquet.    It  was  supposed  by  Detre  that 
the  tuberculin  reaction  elicited  will  indicate  the  type  of  infection, 
whether  it  be  of  the  bovine  or  the  human  bacillus.    This  test  is  of 
doubtful  value  for  this  purpose. 

(e)  The  Ophthalmo  Reaction  of  Calmette. — One  drop  of  a  1  per  cent, 
dilution  of  old  tuberculin  purified  by  precipitation  with  alcohol  is 
instilled  in  the  conjunctival  sac.    In  tuberculous  subjects  this  instil- 

1  The  von  Pirquet  reaction  is  usually  negative  in,  tuberculous  children  during  the 
acute  stage  of  measles,  and  occasionally  in  whooping-cough. 


DIAGNOSIS  OF  TUBERCULOSIS  451 

lation  is  followed  by  a  reddening  of  the  caruncle  and  usually  the 
conjunctiva  as  well.  The  reaction  varies  in  intensity  from  a  very 
mild  local  reddening  of  the  caruncle  to  a  conjunctivitis.  The  reac- 
tion becomes  visible  usually  in  from  four  to  eight  hours.  The  maxi- 
mum intensity  is  reached  about  the  twelfth  hour  and  it  usually  disap- 
pears within  twenty-four  to  forty-eight  hours.  If  the  first  test  is 
negative  and  it  is  desirable  to  repeat  it  with  a  2  to  4  per  cent,  con- 
centration of  tuberculin,  the  second  instillation  must  be  made  in  the 
unused  eye.  The  eye  first  used  is  sensitized  by  the  first  instillation 
and  will  react  in  a  severe  manner  even  if  the  patient  has  no  tuber- 
culosis.1 This  reaction,  however,  can  only  be  elicited  after  ten  to 
fourteen  days  following  the  first  instillation. 

If  tuberculin  treatment  is  to  be  instituted,  the  ophthalmo  reaction 
is  not  indicated,  for  a  reaction  is  almost  certain  to  take  place  when 
tuberculin  treatment  is  established.  The  ophthalmo  reaction  should 
not  be  used  in  old  people  or  in  individuals  having  other  than  perfectly 
normal  eyes. 

5.  Specificity  of  the  Tuberculin  Reaction. — The  tuberculin  reaction 
is  not  absolutely  specific  as  an  index  of  the  occurrence  within  the  host 
of  an  active  tuberculous  focus.  From  30  to  60  per  cent,  of  adults 
known  -to  contain  no  clinically  active  foci  of  tuberculosis,  who  come 
to  autopsy,  show  evidences  of  healed  tubercles.  In  these  individuals 
the  tubercle  bacillus  has  been  dissolved  and  its  products  have  sen- 
sitized them.  The  tuberculin' reaction,  consequently,  will  be  positive 
in  the  majority  of  these  individuals  because  they  are  sensitized  to 
the  proteins  of  the  tubercle  bacillus.  Young  children  are  much  less 
likely  to  possess  these  healed  or  latent  tuberculous  foci,  and  provided 
they  are  not  too  young,  or  that  they  are  not  born  of  tuberculous 
mothers,  a  positive  tuberculin  reaction  is  much  more  conclusive  in 
them.  Individuals  having  advanced  tuberculous  lesions  occasionally 
do  not  give  a  tuberculin  reaction. 

Miiller2  has  studied  the  von  Pirquet  reaction  in  young  children. 
His  results  follow: 

0-  3  months 8.1  per  cent,  cases  with  positive  reaction 

3-  6  months 7.0  per  cent,  cases  with  positive  reaction 

6—12  months 11.7  per  cent,  cases  with  positive  reaction 

12-24  months 24 . 4  per  cent,  cases  with  positive  reaction 

In  8  cases  with  a  positive  reaction  which  came  to  autopsy  all  were 
found  to  be  tuberculous;  46  negative  cases  which  came  to  autopsy 

1  Rosenau  and  Anderson,  Jour.  Am.  Med.  Assn.,  1908,.!,  961. 

2  Arch.  f.  Kinderheilk.,  1,  18. 


452  THE  TUBERCLE  BACILLUS  GROUP 

were  all  free  from  tuberculosis,  except  one  which  had  subsequently 
developed  miliary  tuberculosis.  His  general  conclusion  is  that  the 
unreliability  of  this  reaction  increases  with  age. 

Von  Ruck1  has  collected  a  large  series  of  cases  from  the  literature 
with  the  following  results : 

Subcutaneous  reaction,  8108  cases  in  all. 

4803  tuberculous  patients  (clinical)        ....  4318  positive  reaction 

485  suspected  cases  (clinical)     .      .      .      .      .      .  318  positive  reaction 

2820  clinically  non-tuberculous 1444  positive  reaction 

Cutaneous  reaction,  6571  cases. 

2192  tuberculous  patients  (clinical)        .      .      .      .  1851  positive  reaction 

865  suspected  cases  (clinical) 563  positive  reaction 

3514  clinically  non-tuberculous 1047  positive  reaction 

Conjunctival  reaction,  6788  cases. 

2834  tuberculous  patients  (clinical)        ....  2370  positive  reaction 

1188  suspected  cases  (clinical) 685  positive  reaction 

2766  clinically  non-tuberculous 407  positive  reaction 

SUMMARY. 

Tuberculosis  Suspicious       Non-tuberculosis 

Subcutaneous  reaction        .      .     89.2%+  65.5%+         51.2%  + 

Conjunctival  reaction  .      .      .     83. 6%+  56. 6%+         14.7  %  + 

Cutaneous  reaction        .      .      .     84.4  %  +  65.0  %  +         29.8  %  + 
Cutaneous  reaction  (excluding 

children) 33.9  %  + 

The  tuberculin  test,  therefore,  when  positive  gives  no  absolutely 
definite  distinction  between  healed,  latent,  or  active  foci  of  infection 
with  the  tubercle  bacillus.  Furthermore,  no  quantitative  evidence 
of  the  nature  of  the  reaction  or  extent  of  the  lesions  is  elicited  by  a 
positive  reaction.  A  negative  reaction  properly  performed,  however, 
in  non-cachectic  subjects  or  those  who  have  not  had  progressive 
treatment  with  tuberculin  is  fairly  conclusive.  The  subcutaneous 
test  of  Koch  is  moderately  reliable  in  adults  provided  the  dosage  is 
correctly  selected — a  matter  requiring  unusual  skill  and  experience. 
The  von  Pirquet  reaction  is  quite  unreliable  as  an  index  of  an  active 
focus  in  adults,  because  from  30  to  60  per  cent,  of  all  persons  over 
five  years  of  age  react  positively  in  varying  degrees.  It  is  a  fairly  con- 
clusive test  in  children  under  five  years  of  age.  Similar  fallacies  are 
to  be  expected  in  the  ophthalmo  and  Moro  reactions.2 

B.  Serological  Diagnosis. — 1.  Opsonic  Index. — Wright  and  his  pupils 
have  followed  the  opsonic  index  in  tuberculous  patients  and  they 

1  Beitr.  z.  Klinik  d.  Tuberkulose,  1909,  xiii,  Heft  1. 

2  For  a  detailed  summary  of  the  Koch,  Wassermann,  von  Pirquet,  and  Wolff-Eisner 
theories  of  the  tuberculin  reaction  and  its  importance  for  therapy,  see  Kuthy  and  Wolff- 
Eisner,   Die  Prognosenstellung  bei  den  Lungentuberkulose,  Berlin  and  Vienna,   1914, 
393-399.     Also,  Wolff-Eisner,  Die  Tuberkulinbehandlung,  1913. 


DIAGNOSIS  OF  TUBERCULOSIS  453 

believe  that  the  changes  in  opsonic  index  furnish  a  reliable  index 
for  treatment  with  tuberculin  or  other  products  of  the  tubercle  bacillus. 
The  inherent  and  unavoidable  errors  of  the  opsonic  index  determina- 
tion make  it  unreliable  for  general  use.1 

2.  Agglutination. — Agglutinins   occur   in  tuberculous   patients,2  but 
the  agglutinating  reaction  is  unreliable,  partly  because  of  the  diffi- 
culty in   obtaining   a   proper  suspension    of   tubercle  bacilli.      It   is 
practically  never  used  in  practice.3 

3.  Complement  Fixation. — Practically  never  used  at  the  present  time.4 
C.  Bacteriological    Diagnosis. — 1.   Principle  Involved. — (a)  Micro- 
scopical Examination. — Fluids,  tissues  or  exudates  suspected  to  con- 
tain tubercle  bacilli  are  stained  preferably  by  the  Ziehl-Neelsen  method 
for  the  demonstration  of  acid-fast  bacilli  having  the  morphology  of 
the  tubercle  bacillus.     Other  acid-fast  bacilli  may  be  confused  with 
the  tubercle  bacillus  and  their  presence  must  be  borne  in  mind  when 
microscopical  examinations  are  made.    These  will  be  discussed  under 
their  appropriate  headings. 

It  is  essential  to  use  absolutely  new  slides  for  examination  of  the 
tubercle  bacillus;  old  slides  which  have  been  used  for  this  purpose 
not  infrequently  retain  tubercle  bacilli.  It  is  also  advisable  not  to 
make  a  positive  diagnosis  unless  ten  typical  tubercle  bacilli  can  be 
demonstrated  in  the  preparation.  Among  thousands  of  smears  which 
have  been  examined  by  Arms,  formerly  of  the  Boston  State  Board  of 
Health  Laboratory,  only  one  has  failed  to  show  ten  tubercle  bacilli, 
if  any  were  present,  after  careful  search.  This  one  case  was  shown 
by  many  subsequent  examinations  to  be  negative  for  tubercle  bacilli. 
The  microscopical  examination  is  the  most  rapid  method  of  diagnosing 
tuberculosis  bacteriologically. 

(b)  Cultural. — Cultures  of  typical  bacilli  mav  be  obtained  either 
directly  from  lesions,  fluids,  tissues,  or  exudates,  or  from  animals 
following  inoculation  with  suspected  material.  In  order  to  obtain  cul- 
tures of  tubercle  bacilli  from  material  containing  other  organisms  as  well, 
it  is  necessary  to  treat  the  material  first  with  antiformin5  from  two 

1Trudeau,  Am.  Jour.  Med.  Sc.,  1907,  cxxxiii,  813;  Baldwin,  New  York  Mcd.  Jour., 
June  27,  1908. 

2  Romberg,  Deutsch.  med.  Wchnschr.,  1901,  275,  295. 

3  Eisenberg  and  Keller,  Centralbl.  f.  Bakt.,  1903,  xxxiii,  549,  for  literature. 

4  Stimpson,  Bull.  101,  Hygienic  Laboratory,  1915,  for  literature. 

5  Paterson,  Antiformin  for  the  Detection  of  Tubercle  Bacilli  in  Sputum.     Studies  from 
the  Saranac  Laboratory  for  the  Study  of  Tuberculosis,  1904-1910.    Sodium  carbonate, 
600  grams;   fresh  chlorinated  lime,  400  grams;   distilled  water,  4000  grams.     Dissolve 
the  sodium  carbonate  in  1000  c.c.  distilled  water;   triturate  the  chlorinated  lime  in  3000 
c.c.  distilled  water;   filter,  and  mix  with  the  sodium  carbonate  solution.     Filter  again. 


454  THE  TUBERCLE  BACILLUS  GROUP 

to  three  hours  so  as  to  kill  off  the  contaminating  bacteria.  Anti- 
formin  does  not  as  a  rule  kill  tubercle  bacilli  during  this  time.  It 
is  necessary  to  remove  the  antiformin  by  repeated  washing  and  cen- 
trifugalization  of  the  tubercle  bacilli  before  the  latter  are  inoculated 
into  artificial  media,  preferably  Dorset's  egg  medium.  At  best  the 
cultural  procedure  is  an  unsatisfactory  one. 

(c)  Animal  Inoculation. — Animal  inoculation  is  the  most  delicate 
test  for  demonstrating  the  presence  of  tubercle  bacilli.  Guinea-pigs 
are  the  animals  selected,  and  the  method  of  inoculation  depends  upon 
the  nature  of  the  material.  If  the  material  is  suspected  to  contain 
tubercle  bacilli  only,  it  is  introduced  directly  under  the  skin,  or,  better, 
intraperitoneally.  If  other  organisms  are  associated  with  the  tubercle 
bacillus,  the  material  mav  either  be  mixed  with  antiformin  and  shaken 
for  two  hours  to  kill  off  or  weaken  the  other  organisms,  then  washed 
to  remove  the  antiformin  and  the  sediment  injected,  or  the  material 
may  be  introduced,  contaminating  organisms  and  all,  subcutaneouslv 
into  a  guinea-pig  in  the  following  manner.  A  subcutaneous  pocket 
is  made  on  the  flank  of  the  guinea-pig  and  the  suspected  material  is 
introduced  beneath  the  skin  and  pushed  forward.  The  cut  is  left 
open  and  whatever  pus-producing  organisms  are  present  cause  sup- 
puration; the  pus  drains  away  and  the  initial  inflammation  is 
recovered  from  before  the  tubercle  bacilli  kill  the  animal.  Tubercle 
bacilli  then  may  be  recovered  from  the  regional  lymph  nodes  and  the 
internal  organs.  When  the  inguinal  glands  are  well  enlarged  the 
animal  is  chloroformed.  The  skin  is  sterilized  with  bichloride  of  mer- 
cury and  sterile  instruments  are  used  in  performing  the  autopsy. 
Bits  of  tissue  from  the  lymph  glands,  spleen,  or  other  organs  are 
removed  aseptically  and  dropped  on  the  surface  of  specially-prepared 
media,  preferably  the  Dorset  egg  medium.  A  microscopical  examina- 
tion is  also  made  in  the  usual  manner. 

Sputum. — It  should  be  remembered  that  apparently  normal  indi- 
viduals may  infrequently  have  acid-fast  bacilli  in  their  sputum  and, 
rarely,  tubercle  bacilli,  without  producing  apparent  symptoms. 
Sputum  from  suspected  tuberculous  patients  may  be  examined  directly 
by  stained  smears,  in  which  case  the  early  morning  sputum  coming 
from  the  depths  of  the  lungs  is  to  be  employed.  The  caseous  or  puru- 
lent masses  which  are  characteristic  of  tubercular  sputum  are  removed, 
spread  upon  slides  and  examined  after  staining  with  carbol-fuchsin 
and  decolorizing  in  the  usual  manner.  If  the  result  is  negative,  the 
sputum  may  be  mixed  with  caustic  soda  or  antiformin,  shaken,  and 


DIAGNOSIS  OF  TUBERCULOSIS  455 

the  sediment  examined  by  staining  with  carbol-fuchsin.  If  this 
method  proves  negative,  cultures  from  the  sputum  may  be  made 
after  treating  it  with  antiformin  for  two  to  three  hours.  Some  of  the 
sediment,  after  washing  to  remove  the  antiformin,  should  be  injected 
into  guinea-pigs.  It  is  usually  difficult  to  find  tubercle  bacilli  in  the 
sputum  during  hemoptysis,  but  they  are  found  very  frequently  in  the 
blood-streaked  sputum  following  hemoptysis. 

Blood. — Tubercle  bacilli  are  usually  not  found  in  the  peripheral 
blood.  If  they  do  occur  they  are  there  almost  invariably  in  very  small 
numbers.  Occasionally  positive  results  have  been  reported  in  examina- 
tions of  stained  preparations  made  directly  from  the  blood  when  every 
precaution  has  been  taken  to  preclude  the  inclusion  of  extraneous 
acid-fast  organisms.  It  is  far  more  satisfactory,  however,  to  inoculate 
the  blood  subcutaneously  into  guinea-pigs. 

The  Nasal  Cavity. — Tubercle  bacilli  have  been  found  occasionally 
in  the  nasal  passages  of  healthy  individuals,  particularly  those  who 
have  been  closely  in  association  with  tuberculous  patients.  It  must 
be  remembered  that  the  acid-fast  organism  described  by  Karlinsky 
as  the  "nasal  secretion  bacillus,"  is  found  occasionally  both  in  the 
noses  of  tuberculous  patients  and  in  healthy  individuals.  This 
organism  grows  readily  on  artificial  media  and  should  not  be  confused 
with  the  true  tubercle  bacillus. 

Pus  and  Exudates. — It  is  frequently  difficult  or  impossible  to 
detect  tubercle  bacilli  in  the  pus  of  cold  abscesses.  Material  derived 
from  this  source  is  very  frequently  caseous,  and  although  tubercle 
bacilli  can  not  be  demonstrated  by  staining  methods,  the  so-called 
Much  granules  are  found  occasionallv,  which  are  Gram-positive  but 
not  acid-fast.  In  order  to  demonstrate  the  infectiousness  of  this 
material  it  is  inoculated  into  guinea-pigs.  Tubercle  bacilli  may  be 
found  quite  frequently  in  exudates,  but  in  order  to  make  the  diagnosis 
reliable  the  material  should  be  inoculated  into  guinea-pigs. 

Urine. — As  a  rule  it  is  difficult  to  find  tubercle  bacilli  in  urine,  and 
the  probability  of  contamination  with  the  smegma  bacillus  or  the 
Lustgarten  bacillus  must  constantly  be  borne  in  mind.  Sediment  ing- 
large  volumes  of  urine  and  injecting  the  sediment  into  guinea-pigs 
is  the  most  practical  method  of  detecting  tubercle  bacilli  in  this 
excretion,  for  it  rules  out  both  the  Lustgarten  and  smegma  bacilli, 
which  do  not  produce  lesions  in  guinea-pigs.  The  slight  differences 
in  acid-  and  alcohol-fastness  between  these  organisms  and  the  tubercle 
bacillus  make  diagnosis  by  the  direct  smear  method  of  doubtful  value. 


456  THE  TUBERCLE  BACILLUS  GROUP 

Feces. — Tubercle  bacilli  may  appear  in  the  feces  either  because  they 
have  been  swallowed  with  the  sputum  or  because  of  the  existence  of 
tuberculous  ulcers  in  the  intestinal  tract.1  Acid-fast  bacilli  which  are 
not  true  tubercle  bacilli  are  quite  common  in  the  feces,  and  for  this 
reason  animal  inoculation  after  treatment  of  the  feces  with  antifonnin 
is  the  best  method  for  demonstrating  the  organism. 

Milk. — Tubercle  bacilli  are  very  infrequent  in  human  milk,  although 
they  are  said  to  be  relatively  common  in  unpasteurized  cow's  milk 
as  it  is  sold  in  large  cities.  The  organisms  get  into  the  cow's  milk  far 
more  frequently  through  the  contamination  with  feces  than  from 
direct  infection  through  the  udder.  Microscopical  examination  of 
the  sediment  of  milk  or  cream  is  usually  valueless,  as  is  the  examina- 
tion of  the  cream  layer  itself.  Acid-fast  bacilli  which  are  not  tubercle 
bacilli  very  frequently  cause  confusion.  Among  these  organisms  are 
those  described  by  Petri  and  Rabinovitch,  which  are  called  butter 
bacilli.  Inoculation  of  the  sediment  and  of  the  cream  of  milk  into 
guinea-pigs  is  the  only  safe  test. 

Immunity  and  Immunization.2 — The  disproportion  between  the  inci- 
dence of  "healed  tubercles"  in  cadavers  which  do  not  exhibit  symptoms 
of  tuberculosis  ante  mortem  and  the  actual  number  of  clinical  cases 
suggests  that  the  average  individual  possesses  a  certain  degree  of 
refractoriness  to  progressive  invasion  by  the  tubercle  bacillus,  that 
is  to  say,  the  clinical  cases  of  the  disease  are  considerably  outnumbered 
by  those  in  whom  the  organism  has  gained  entrance,  but  failed  to 
develop  sufficiently  to  cause  symptoms.  Early,  uncomplicated  cases 
of  tuberculosis  frequently  react  favorably  when  placed  in  a  favorable 
environment.  Spontaneous  recovery  from  tuberculosis  complicated 
by  secondary  infections  with  other  bacteria  is  more  tedious  and  the 
prognosis  is  generally  less  favorable. 

Active  immunization  of  man  with  various  products  of  the  tubercle 
bacillus  has  been  one  of  the  greatest  problems  of  medicine.  Up  to 
the  present  time  the  solution  of  this  problem  has  not  been  realized. 
At  least  a  generation  must  elapse  before  final  judgment  can  be  passed 
upon  any  system  of  human  immunization,  for  the  disease  tuberculosis 
progresses  slowly  and  results  to  be  trustworthy  must  be  numerous  and 
of  long  duration. 

1  Laird,  Kite,  and  Stewart,  Jour.  Med.  Research,  1913,  x'xix,  31,  for  summary  and 
literature. 

2  For  an  excellent  summary  of  Immunity  in  Tuberculosis,  see  Baldwin,  Am.  Jour.  Med. 
Sc.,  1915,  cxlix,  822. 


DIAGNOSIS  OF  TUBERCULOSIS  457 

Active  immunization  has  been  attempted  with  the  following  types 
of  preparation : 

1.  Tuberculins. 

2.  Vaccines. 

(a)  Killed  cultures. 
(6)  Soluble  vaccines. 

3.  Living  tubercle  bacilli. 

(a)  Virulent  organisms,  Webb  method. 
(6)  Attenuated  viruses, 
(c)  Alien  acid-fast  bacilli. 

4.  Sera. 

It  has  long  been  recognized  that  various  tuberculins  do  not  confer 
immunity  upon  experimental  animals,  although  in  skilled  hands  they 
possess  undoubted  curative  value.1  Killed  cultures  of  tubercle  bacilli 
have  not  been  satisfactory,  and  their  use  has  been  greatly  restricted 
by  the  non-solubility  of  the  organisms  which  produce  local  indurations 
of  refractory  nature.  Soluble  vaccines  including  "bacillus  emulsions" 
and  various  proteins  of  the  bacillus  do  not  appear  to  confer  definite 
immunity  upon  susceptible  animals.  The  superiority  of  living  viruses 
over  the  various  preparations  of  killed  organisms  and  their  products 
for  protective  inoculation  is  conceded  by  the  great  majority  of  inves- 
tigators. Webb  and  Williams2  have  attempted  to  induce  artificial 
active  immunity  in  experimental. animals  by  injecting  virulent  tubercle 
bacilli,  beginning  with  one  or  two  organisms  and  gradually  increasing 
the  number.  Their  results,  while  few  in  number,  appear  to  be  worthy 
of  serious  consideration.  The  use  of  attenuated  cultures  and  of  alien 
acid-fast  bacilli  have  not  been  successful  up  to  the  present  time. 
Sera  have  been  equally  unsatisfactory. 

Bovine  Tubercle  Bacillus. — Cattle  and  swine  are  susceptible  to 
infection  with  the  bovine  tubercle  bacillus  and  the  disease  is  widespread 
among  dairy  herds.  Statistics  indicate  that  in  certain  parts  of  the 
United  States  the  incidence  of  tuberculosis  in  swine  increases  almost 
proportionately  to  the  spread  of  the  disease  among  cattle.  This 
condition  is  brought  about  partly  through  the  practice  of  feeding 
slaughter-house  offal  to  swine,  chiefly  through  dairies  and  cheese 
factories  where  the  skimmed  milk  or  whey  forms  a  not  inconsiderable 
part  of  the  rations  of  swine.  Scrofula  or  tuberculosis  of  swine  thus  is 
a  true  ingestion  disease.  It  is  possible  to  infect  swine  by  feeding  human 

1  Trudeau,  Osier's  Modern  Medicine,   1907,  iii,  434. 

2  Jour.  Med.  Research,  1909,  xx,  1;    1911,  xxiv,  1. 


458  THE   TUBERCLE  BACILLUS  GROUP 

tubercle  bacilli;  the  spontaneous  disease,  however,  is  almost  invariably 
an  infection  with  the  bovine  type  of  the  tubercle  bacillus.1 

The  most  common  initial  lesion  in  cattle  is  an  involvement  of  the 
retropharyngeal  glands;  the  lungs  are  frequently  infected,  and  occa- 
sionally the  liver  and  serous  membranes  are  invaded  rather  early  in 
the  disease.  A  peculiar  and  characteristic  type  of  infection  of  cattle, 
known  as  Perlsucht  or  pearly  disease,  which  progresses  slowly  and  is 
recognizable  only  in  the  later  stages,  is  distinguished  by  the  occurrence 
upon  serous  surfaces  of  thick  fibrous  tumors  containing  much  con- 
nective tissue.  The  lesions  in  infections  of  the  peritoneal  surfaces 
consist  of  large  numbers  of  solitary  or  clustered  tubercles  varying  in 
size  from  1  to  more  than  10  mm.  in  diameter.  They  may  be  attached 
to  the  surface  by  tough,  fibrous  pedicles  or  they  may  rest  directly  upon 
the  membrane  itself.  These  tumors  may  become  calcified  or  caseated 
and  they  are  larger  than  tubercles  found  in  human  tissues.  Morpho- 
logically their  structure  is  fundamentally  not  unlike  human  tubercles. 

Theobald  Smith2  was  the  first  to  clearly  point  out  the  differences 
between  the  human  and  bovine  tubercle  bacilli.  His  evidence  was 
based  upon  morphological,  cultural  and  pathological  characters.  He 
showed  that  the  human  tubercle  bacillus,  grown  on  serum,  was  longer 
and  slenderer  than  the  bovine  type  and  frequently  curved.  The 
growth  is  more  luxuriant,  forming  a  thick,  wrinkled  membrane  upon 
glycerin  broth.  The  bovine  type  commonly  develops  feebly  in  this 
medium  and  produces  a  thin,  delicate  pellicle.  The  reaction  curves 
of  the  two  types  on  glycerin  broth  are  distinctive  and  characteristic. 
The  bovine  type  gradually  creates  an  alkaline  reaction;  the  human 
type  leaves  the  reaction  acid  and  tuberculin  made  with  the  bovine 
type  consequently  is  alkaline  in  reaction;  that  of  the  human  type  is 
acid.  The  pathogenic  action  of  the  two  types  is  distinctive;  the 
bovine  type  is  highly  pathogenic  for  rabbits  and  calves;  the  human 
type  is  only  slightly  pathogenic  for  these  animals.  One  milligram  of 
a  human  culture  fails  to  kill  rabbits,  but  0.1  milligram  of  a  freshly 
isolated  bovine  culture  results  fatally. 

The  important  differential  characters  are  summarized  in  the  follow- 
ing table: 

HUMAN  TYPES.  BOVINE  TYPES. 

MORPHOLOGY. 

On  serum    or  glycerin    bouillon,  long,  On   serum   or    glycerin   bouillon,    rela- 

slender,  slightly  curved  rods  which  lively  short  thick  rods  irregularly  ar- 
usually  stain  uniformly;  occur  in  clusters  -  ranged;  frequently  exhibit  slight  irregu- 
usually  lying  parallel.  larity  in  staining. 

1  Theobald  Smith,  Boston  Medical  and  Surgical  Journal,  1909,  clix.  707. 

2  Jour.  Exp.  Med.,  1898,  iii,  451. 


DIAGNOSIS  OF   TUBERCULOSIS  459 

CULTURAL    CHARACTERS. 

Glycerin    bouillon,    after   two    to    four  Glycerin   bouillon,   delicate  membrane 

weeks'  growth,  dense  wrinkled  membrane.  exhibiting    occasional    wrinkling    of    the 

surface. 

Reaction  remains  permanently  acid.  Reaction  gradually  becomes  alkaline. 

Tuberculin  has  acid  reaction.  Tuberculin  has  alkaline  reaction. 

Growth     on     blood     serum    relatively  Growth    on     blood     serum     relatively 

luxuriant  and  develops  with  comparative  meagre — develops  slowly, 
rapidity. 

ANIMAL    PATHOGENESIS. 

Guinea-pigs  very  susceptible.     Young  Guinea-pigs,  young  cattle,  rabbits  and 

cattle,    rabbits    and    swine    resistant    to         swine  very  susceptible  to  infection, 
infection. 

Bovine  infections  in  man  are  much  more  common  in  children  than 
in  adults1  Milk  is  a  frequent  vehicle  for  the  transmission  of  the  virus 
to  man;  the  origin  of  the  bacilli  in  milk  has  been  summarized  by 
Moore2  as  follows: 

"1.  Cows  with  tuberculous  udders  eliminate  tubercle  bacilli  with 
the  milk.  In  such  cases  these  organisms  are  usually  present  in  large 
numbers. 

2.  Cows  with  glandular  or  pulmonary  tuberculosis,  in  which  the 
lesions  are  discharging  into  the  bronchi,  eliminate  tubercle  bacilli 
with  the  feces  and  with  the  droolings.    In  cases  of  intestinal  tuberculous 
ulcers  the  organisms  are  excreted  with  the  feces. 

3.  Milk  is  usually  infected  with  tubercle  bacilli  when  it  is  taken 
from  cows  with  tuberculous  udders.     It  may,  through  contamination 
with  feces  or  uterine  discharges,  be  infected  when  drawn  from  cows 
with  open  lesions  in  the  respiratory  and  digestive  tracts  or  organs  of 
reproduction. 

4.  Tubercle  bacilli  are  not,  as  a  rule,  present  in  milk  of  cows  that 
react  to  tuberculin  and  which,  on  careful  physical  examination,  exhibit 
no  evidence  of  disease." 

The  identification  of  tubercle  bacilli  in  milk  presents  no  insurmount- 
able difficulties,  but  certain  precautions  must  be  observed.  Prudden 
and  Hodenpyl,3  Straus  and  Gamaleia4  and  others  have  shown  that  the 
injection  of  killed  tubercle  bacilli  into  guinea-pigs  by  the  intraperi- 
toneal  route  will  induce  tubercle  formation  even  if  the  organisms  have 
been  heated  in  the  autoclave.  These  tubercles,  however,  if  crushed 
and  injected  into  fresh  animals,  do  not  reproduce  tubercles.  It  is 
obvious  that  the  injection  of  pasteurized  milk  containing  dead  tubercle 

1  Statistics  by  Park  and  Krumwiede,  p.  438. 

2  Jour.  Med.  Research,  1911,  xxiv,  517. 

3  New  York  Med.  Jour.,  1891,  liii,  637,  697. 
*  Arch.  med.  exper.,  1891,  iii,  705. 


460  THE  TUBERCLE  BACILLUS  GROUP 

bacilli  may  lead  to  false  conclusions  unless  this  possibility  be  borne  in 
mind.  The  bacillus  of  infectious  abortion  induces  lesions  in  guinea- 
pigs  closely  simulating  tuberculosis.1  This  organism  is  rather  widely 
distributed  in  unheated  milk.2 

Method. — Ten  to  20  c.c.  of  milk  are  centrifuged  for  half  an  hour 
and  from  5  to  10  c.c.  of  the  sediment  and  lower  portion  of  the  sample 
are  injected  subcutaneously  into  guinea-pigs.  The  cream  layer  is 
also  injected  subcutaneously  into  a  second  pig.  After  three  to  six 
weeks,  if  the  animal  shows  signs  of  emaciation,  0.5  cm.  of  undiluted 
bovine  tuberculin  is  injected.  This  injection  usually  results  fatally. 
In  any  event  the  animal  is  killed  and  the  bovine  tubercle  bacilli  are 
identified  in  the  usual  manner. 

Immunity. — Cattle  and  swine  exhibit  little  or  no  natural  immunity 
to  infection  with  the  bovine  tubercle  bacillus.  The  most  satisfactory 
prophylaxis  consists  in  isolating  infected  animals  from  the  herd, 
disinfection  of  the  stables  and  testing  all  apparently  sound  animals 
with  tuberculin. 

Tuberculin  Test. — The  preparation  of  bovine  tuberculin  is  precisely 
similar  to  that  of  human  tuberculin,  except  that  the  bovine  organism 
is  used.  The  test  is  carried  out  in  the  following  manner: 

The  temperature  of  the  animal  is  taken  at  frequent  intervals  for 
twenty-four  hours,  then  tuberculin  is  injected  subcutaneously,  prefer- 
ably over  the  fore-shoulder,  about  10.00  P.M.  Temperatures  are  taken 
from  6.00  A.M.  of  the  following  morning  at  two-hour  intervals  until 
10  P.M.  An  elevation  "of  temperature  of  from  one  to  three  degrees 
occurs  within  a  few  hours  in  positive  cases  and  a  hot  swollen  area  of 
induration  appears  around  the  site  of  inoculation.  Both  the  febrile 
reaction  and  the  indurated  area  slowly  become  normal.  The  Inter- 
national Commission  on  the  Control  of  Bovine  Tuberculosis3  states: 

"1.  That  tuberculin,  properly  used,  is  an  accurate  and  reliable 
diagnostic  agent  for  the  detection  of  active  tuberculosis. 

2.  That  tuberculin  may  not  produce  a  reaction  under  the  following 
conditions : 

(a)  When  the  disease  is  in  the  period  of  incubation. 

(6)  When  the  progress  of  the  disease  is  arrested. 

(c)  When  the  disease  is  extensively  generalized. 

1  Theobald  Smith,  Bureau  of  Animal  Industry,  1894,  Bull.  7,  80     Smith  and  Fabyan, 
Centralbl.  f.  Bakt.,  Orig.,  1912,  Ixi,  549. 

2  Melvin,  Vet.  Jour.,  1912,  Ixviii,  528.     Fabyan,  Jour.  Med.  Research,  1913,  xxviii,  85. 

3  Forty-seventh  Annual  Report  of  the  American  Veterinary   Medical  Association, 
September,  1910. 


DIAGNOSIS  OF  TUBERCULOSIS  461 

The  last  condition  is  relatively  rare  and  may  usually  be  detected 
by  physical  examination. 

3.  On  account  of  the  period  of  incubation  and  the  fact  that  arrested 
cases  may  sooner  or  later  become  active,  all  exposed  animals  should 
be  retested  at  intervals  of  six  months  to  one  year. 

4.  That  the  tuberculin  test  should  not  be  applied  to  any  animal 
having  a  temperature  higher  than  normal. 

5.  That  an  animal  having  given  one  distinct  reaction  to  tuberculin 
should  thereafter  be  regarded  as  tuberculous. 

6.  That  the  subcutaneous  injection  of  tuberculin  is  the  only  method 
of  using  tuberculin  for  the  detection  of  tuberculosis  in  cattle,  which 
can  be  recommended  at  the  present  time. 

7.  That  tuberculin  has  no  injurious  effect  upon  healthy  cattle." 
Avian   Tubercle   Bacillus. — Hens,  pheasants  and  other   birds   are 

subject  to  a  spontaneous  disease  which  is  anatomically  very  much 
like  tuberculosis  of  other  warm-blooded  animals.  Koch1  believed 
that  the  organism  of  avian  tuberculosis  was  identical  with  the  bovine 
tubercle  bacillus,  but  later  work  has  not  confirmed  this  assertion. 
It  is  believed  at  the  present  time  that  the  bovine  and  avian  tubercle 
bacilli  are  distinct  entities. 

The  morphology  of  the  avian  tubercle  bacillus  and  its  staining 
reactions  are  quite  similar  to  those  of  the  bovine  organism,  except 
that  pleiomorphism  is  more  marked  in  the  former,  particularly  when 
it  is  grown  at  40°  to  42°  C.  It  forms  no  spores  and  no  capsules,  is 
non-motile  and  has  no  flagella.  It  grows  more  readily  than  either 
the  human  or  the  bovine  strains;  the  addition  of  glycerin  to  media, 
while  not  essential,  increases  the  luxuriance  of  the  growth.  On  coagu- 
lated blood  serum  or  agar  after  six  to  ten  days  the  organisms  appear 
as  small  white  colonies  with  a  waxy  luster.  A  second  transfer  to  arti- 
ficial media  results  in  a  more  luxuriant  growth  which  spreads  and 
increases  in  luxuriance,  eventually  covering  the  whole  medium.  The 
growth  is  moist  and  may  become  slimy,  differing  markedly  in  this 
respect  from  the  human  and  bovine  types.  The  pellicle  formed  on 
broth  cultures  is  less  friable  and  more  tenacious  than  that  characteris- 
tic of  the  mammalian  strains.  The  range  of  growth  is  from  35  to  45 
degrees;  40°  is  the  optimum  temperature,  but  development  is  luxu- 
riant at  37°  C.  An  exposure  of  two  hours  at  65°  C.  usually  fails  to 
kill  avian  tubercle  bacilli— but  fifteen  minutes  at  70°  to  72°  C.  is 
fatal.  The  organisms  are  very  resistant  to  drying,  remaining  alive 

1  Mitt.  a.  d.  Kais.  Ges.  Amte,  1884,  ii,  4. 


462  THE   TUBERCLE  BACILLUS  GROUP 

for  several  months  both  in  cultures  and  in  uncontaminated  material 
from  infected  birds.  Unlike  the  human  or  the  bovine  disease,  avian 
tuberculosis  is  transmitted  ordinarily  as  a  congenital  infection.  Birds 
are  comparatively  readily  infected  artificially,  however,  by  injection 
of  the  organisms.  Edwards1  has  infected  hens  with  the  excrement  of 
infected  birds.  The  liver  and  the  spleen  are  the  organs  more  commonly 
involved.2 

Among  laboratory  animals  rabbits  appear  to  be  more  susceptible 
than  guinea-pigs,  although  Edwards3  appears  to  have  successfully 
infected  several  guinea-pigs  with  pure  cultures  of  the  organism. 
Moore4  was  unsuccessful  in  causing  the  bacilli  to  multiply  in  guinea- 
pigs  but  observed  that  the  animals  frequently  died  of  marasmus, 
apparently  from  the  absorption  of  toxins  from  the  bacilli;  Mohler5 
has  induced  infection  in  swine  by  feeding  them  the  carcasses  of  tuber- 
culous hens.  Himmelberger6  has  made  the  important  observation 
that  calves  may  be  susceptible  to  infection  with  the  avian  tubercle 
bacillus. 

1  Ont.  Agricult.  College  Bull.,  No.  193. 

2  De  Jong,  Ann.  Inst.  Past.,  1910,  xxiv. 

3  Loc.  cit. 

4  Jour.  Med.  Research,  xi,  521. 

5  Twenty-fourth  Annual  Report,  Bureau  of  Animal  Industry. 

6  Centralbl.  f.  Bakt.,  Orig.,  1914,  Ixxiii,  1. 


CHAPTER  XXIV. 


LEPROSY  AND  ACID-FAST  BACTERIA  OTHER  THAN 
THE  TUBERCLE  GROUP. 


THE  BACILLUS  LEPILE. 
Leprosy  of  Rats. 

ACID-FAST  BACILLI  OTHER  THAN  BA- 
CILLUS TUBERCULOSIS  AND  BACILLUS 
LEPR.E. 


The  Smegma  Bacillus. 

The  Nasal  Secretion  Bacillus. 

Bacillus  Phlei. 

The  Butter  Bacillus. 


THE   BACILLUS   LEPR^I. 

THE  first  definite  observation  of  the  organism  now  called  Bacillus 
leprse  was  that  of  Hansen,1  who  described  rod-shaped  organisms  in 
leprous  tissue.  Somewhat  later  Neisser2  succeeded  in  staining  them. 
Sticker3  made  the  important  discovery  that  the  nasal  mucosa  and 
nasal  secretion  of  a  large  percentage  of  lepers  (140  out  of  a  total  of 
153  cases  examined)  contain  large  numbers  of  leprosy  bacilli.  The 
organism  described  by  Hansen  is  generally  accepted  as  the  causative 
agent  of  leprosy. 

Morphology. — Morphologically  Bacillus  leprse  resembles  Bacillus 
tuberculosis.  It  is  a  slender,  rod-shaped  organism,  measuring  from 
4  to  6  microns  in  length.  Usually  it  is  somewhat  shorter  than  the 
tubercle  bacillus,  curved  forms  are  less  common,  and  the  ends  of  the 
bacilli  are  not  infrequently  somewhat  enlarged.  They  occur  charac- 
teristically as  clusters  of  bacilli,  grouped  together  in  bundles  like 
cigars,  lying  within  large  cells — the  so-called  lepra  cells.  The  organisms 
from  young  lepra  nodules  are  more  acid-fast  than  the  tubercle  bacillus; 
in  old,  degenerating  nodules  they  tend  to  lose  their  acid-fastness  and 
other  staining  properties,  and  the  cytoplasm  becomes  vacuolated, 
giving  the  bacilli  a  beaded  appearance.  Bacillus  leprse  is  Gram-positive 
as  well  as  acid-fast.  Available  evidence  indicates  that  the  organism 
is  non-motile,  possesses  no  flagella,  and  forms  no  capsules. 


1  Virchow's  Arch.,  1880,  Ixxix,  32. 

2  Ibid.,  1881,  Ixxxiv,  514. 

3  Berl.  klin.  Wchnschr.,  1897,  518;   Arb.  a.  d.  kais.  Gesamte,  1899,  xvi,  357. 


464  LEPROSY  AND  ACID-FAST  BACTERIA 

Bacillus  leprse  differs  somewhat  from  Bacillus  tuberculosis  in  its 
staining  reactions  and  occurrence  in  lesions. 

BACILLUS  LEPR^E.  BACILLUS  TUBERCULOSIS. 

1.  Stains   with    aqueous    solutions   of  Does  not  stain  with  these  dyes?, 
basic  anilin  dyes. 

2.  Stains  readily  by  Gram's  method.  Stains  with  difficulty. 

3.  Resists  decolorization  by  the  Ziehl-  Somewhat  more  readily  decolorized. 
Neelsen  stain. 

4.  Large   numbers   of   acid-fast   bacilli  Relatively  fewer  acid-fast  bacilli  found 
occur  within  swollen  cells — lepra  cells.  together  as  a  rule.     No  cells  resembling 

lepra  cells. 

These  differences  are  quantitative  rather  than  qualitative,  how- 
ever, and  can  not  be  individually  relied  upon  to  establish  an  absolute 
differentiation  between  the  two  organisms.  The  most  distinctive 
difference  is  the  "lepra  cell"  with  its  large  number  of  acid-fast  bacilli 
in  groups  with  their  long  axes  arranged  in  parallel. 

Isolation  and  Culture. — Clegg1  cultivated  an  acid-fast  organism  from 
lepers  by  growing  the  organism  upon  agar  symbiotically  with  amebse, 
then  killing  the  amebse  by  an  exposure  to  60°  C.  for  thirty  minutes. 
The  bacilli,  once  acclimatized  to  artificial  media,  grew  readily.  Duval2 
has  also  cultivated  acid-fast  bacilli  directly  from  lepers.  Animal 
experimentation  and  serological  studies  have  been  inconclusive  thus 
far.  Kedrowski3  described  a  pleiomorphic  streptothrix-like  organism 
which  grew  in  artificial  media  as  a  non-acid-fast  streptothrix,  but 
tended  to  change  to  a  pleiomorphic,  diphtheroid,  acid-fast  bacillus. 
(Plate  III.)  When  this  diphtheroid  bacillus  is  injected  into  rats  or  mice 
it  becomes  acid-fast  and  resembles  Bacillus  leprse  in  detail.  Bay  on4 
states  that  Kedrowski's  organism  is  the  true  leprosy  bacillus,  and  that 
the  diphtheroid  form  is  one  stage  of  the  typical  acid-fast  type  seen 
in  leprous  nodules.  The  entire  subject  of  the  cultivation  of  Bacillus 
leprse  on  artificial  media  must  be  regarded  as  sub  judice. 

Products  of  Growth. — Deycke  and  Reschad5  isolated  a  streptothrix 
(Streptothrix  leproides)  from  a  leper;  from  cultures  of  this  organism 
a  fatty  acid-glycerin  ester  was  prepared,  to  which  the  name  nastin 
was  applied.  Host6  isolated  an  acid-fast  organism  from  lepers,  upon 
salt-free  media.  A  substance,  leprolin,  was  prepared  from  broth  cul- 

1  Philippine  Jour.  Sci.,  1908,  iv,  403. 

2  Jour.  Exp.  Med.,  1910,  xii,  649;    1911,  xiii,  365;    Jour.  Am.  Med.  Assn.,  1912,  Iviii, 
1427. 

3Ztschr.  f.  Hyg.,  1901,  xxxvii,  52. 

4  Centralbl.  f.  Bakt.,  Ref.,  1913,  Ivi,  592. 

8  Deutsch.  med.  Wchnschr.,  1907,  89.     Deycke,  Lepra,  1907,  vii,  174. 

6  British  Med.  Jour.,  1905,  p.  2302. 


PLATE  III 


Lepra  Bacilli;   Ziehl-Neelsen  Stain. 


THE  BACILLUS  LEPR&  465 


tures  of  this  organism  prec^^  as  tuberculin  is  prepared.  Neither 
nastin  nor  leprolin  have  beelr  successful  clinically  judging  from  avail- 
able information. 

Pathogenesis. —  Human. — McCoy  and  Goodhue1  have  summarized 
observations  of  the  infectiousness  of  lepers  in  the  leper  settlement  in 
the  Hawaiian  Islands  as  follows:  of  119  men,  practically  all  Hawaiians 
living  in  the  same  house  with  lepers,  5  (4.40  per  cent.)  developed 
leprosy;  of  106  women,  practically  all  Hawaiians,  living  in  the  same 
house  with  lepers,  5  (4.71  per  cent.)  developed  leprosy;  of  12  women, 
all  Caucasians,  nurses  and  members  of  religious  orders,  living  among 
lepers,  none  contracted  the  disease;  but  of  23  Caucasian  males,  three 
contracted  leprosy.  The  shortest  period  in  which  the  disease  appeared 


FIG.  63. — Lepra  bacilli  in  liver.     (Kolle  and  Hetsch.) 

after  exposure  was  three  years  (2  cases) ;  the  longest  seventeen  years. 
Arning2  inoculated  a  condemned  criminal  with  leprosy  bacilli  derived 
from  a  leper  and  the  criminal  developed  leprosy.  Several  other 
investigators  have  made  similar  experiments,  a  few  of  which  have 
resulted  positively.  The  majority  of  such  attempts  have  been  fail- 
ures, and  the  consensus  of  opinion  is  that  the  few  positive  results  are 
to  be  explained  by  the  existence  of  leprosy  in  the  early  stages. 

The  earliest  lesion  appears  to  be  an  ulcer  at  the  junction  of  the 
bony  and  cartilaginous  septum  of  the  nose.  When  the  bacilli  are  car- 
ried to  any  tissue  in  the  body  they  excite  the  usual  inflammatory 
reaction  which,  however,  is  continued  to  excessive  tissue  proliferation. 
Granular  tissue  containing  bloodvessels  is  formed,  providing  a  good 
vascular  supply,  so  that  proliferation  continues  until  a  fair-sized 

1  Public  Health  Bull.,  1913,  No.  61.  2  Centralbl.  f.  Bakt.,  1889,  v,  672. 

30 


466  LEPROSY  AND  ACID-FAST  BACTERIA 

nodule  or  tumor  is  produced.  If  the  seat  of  infection  is  a  nerve, 
a  spindle-shaped  thickening  is  produced  about  the  nerve,  which 
causes  pressure,  irritation,  inflammation,  degeneration,  and,  finally, 
atrophy,  which  leads  to  anesthesia  of  the  area  of  distribution  of  the 
nerve.  The  bacilli  are  found  both  in  the  neuroglia  and  in  nerve  cells, 
particularly  the  ganglion  cells.  When  the  cell  is  thus  infected  it  under- 
goes degeneration,  sometimes  accompanied  by  swelling  and  formation 
of  vacuoles.  If  the  bacilli  become  attached  to  arteries  a  proliferating 
inflammation  results  which  causes  the  walls  of  the  vessel  to  become 
greatly  thickened  and  the  lumen  narrowed. 

The  disease  often  displays  a  selective  action  for  the  skin,  especially 
in  the  face,  the  extensor  surfaces  of  the  nose  and  elbows,  and  on  the 
backs  of  the  hands  and  feet.  These  areas  are  very  apt  to  undergo 
ulcer  at  ion.  The  organism  makes  its  appearance  in  the  skin  by  pro- 
ducing red  spots  which  either  disappear,  leaving  pigmented  areas,  or 
become  elevated  in  nodules  of  a  brown-red  color.  In  the  region  of 
these  nodules  the  subcutaneous  tissues  contain  large  numbers  of 
bacilli.  These  eruptions  are  probably  to  be  regarded  as  general 
inflammatory  reactions  in  response  to  the  irritation  produced  by  the 
bacillus.  The  nodules  may  remain  small  and  hard,  or  they  may 
become  enlarged,  in  which  case  large  protuberances  appear  which 
destroy  the  symmetry  of  the  face  and  give  the  victim  a  lion-like 
appearance,  hence  the  name  facies  leontina. 

Animal. — It  must  be  remembered  that  tubercle  bacilli  are  very 
frequently  found  in  lepers;  in  fact,  a  not  inconsiderable  number  of 
lepers  die  of  tuberculosis  rather  than  leprosy.  Consequently  it  is 
not  surprising  to  find  that  many  experiments  on  animals  have  resulted 
in  the  production  of  lesions  from  which  acid-fast  bacilli  have  been 
obtained.  These  experiments  must  be  interpreted  with  a  great  deal 
of  caution  for  this  reason.  Nicolle1  claims  to  have  successfully  infected 
a  monkey  (Macacus)  with  leprous  material.  Lesions  appeared  which 
were  nodular  in  character,  but  they  disappeared  spontaneously 
within  six  months.  The  result  is  questionable  and  successful  inocula- 
tions of  leprosy  bacilli  from  lepra  nodules  into  the  lower  animals  are 
not  definitely  proven  as  yet. 

Portal  of  Entry. — Leprous  lesions  appear  early  and  are  fairly  constant 
in  the  nasal  passage,  where  an  ulcer  appears  at  the  junction  of  the 
bony  and  cartilaginous  septum.  This  has  given  rise  to  the  belief 
that  the  nasal  passages  are  the  chief  portals  of  entry  of  the  organism 

1  Semaine  Medicale,  1905,  110. 


THE  BACILLUS  LEPR& 


467 


and  that  infection  takes  place  through  dust  or  droplets.  Recently 
a  case  has  been  described  which  shows  extensive  involvement  of  the 
liver  and  spleen,  in  which  the  intestinal  tract  was  considered  the 
atrium  of  infection.  Cases  also  are  on  record  in  which  the  primary 
lesions  appear  to  have  occurred  on  the  feet,  indicating  that  abrasions 
of  the  skin  may  also  be  portals  of  entry. 

Diagnosis. — Scrapings  from  the  nasal  ulcer  may  give  an  early 
diagnosis.  It  should  be  remembered,  however,  that  an  acid-fast 
organism  described  by  Karlinski  (nasal  secretion  bacillus)  is  fairly 
common,  not  only  in  the  nasal  passages  of  lepers  and  tuberculous 
individuals,  but  also  in  normal  individuals  as  well.  Karlinski's 
organism  grows  readily  on  artificial  media  and  is  in  no  way  related 


d  - 


FIG.  64. — Lepra  bacilli  in  skin.     (Kolle  and  Hetsch.) 

to  the  leprosy  bacillus.  As  a  matter  of  practice,  a  clinical  diagnosis 
of  leprosy  is  more  important  than  a  microscopical  diagnosis;  the 
latter  merely  confirms  the  former.  From  ulcerated  lepromata  or  from 
intact  tuberculoid  nodules  material  may  be  gathered  and  stained  in 
the  usual  manner  for  the  presence  of  acid-fast  organisms.  Inasmuch 
as  the  leprosy  bacilli  occur  in  large  numbers  in  these  tubercles  it  is 
best  to  excise  a  small  portion  of  one,  cut  sections  and  stain  it  for 
leprosy  bacilli.  This  will  give  the  characteristic  arrangement  of  the 
organisms  and  make  the  diagnosis  very  much  more  certain.  Leprosy 
bacilli  can  be  definitely  distinguished  from  tubercle  bacilli;  when 
injected  into  guinea-pigs  they  do  not  produce  lesions. 

Prophylaxis. —  Heredity. — Whether  leprosy  is  a  germinal  infection 
or  not  is  not  known,  although  the  bacilli  have  been  found  both  in 


468  LEPROSY  AND  ACID-FAST  BACTERIA 

ova  and  in  the  testicles.  It  is  suspected  that  children  born  of  leprous 
parents  are  probably  infected  immediately  before  or  shortly  after 
birth. 

Method  of  Dissemination. — The  exact  method  of  transmission  of 
leprosy  is  still  unknown.  It  is  generally  believed  that  the  bacillus 
might  be  transmitted  either  by  droplet  infection  or  by  direct  contact. 
The  organism,  however,  does  not  appear  to  be  very  infectious,  in 
adults  at  least,  for  experience  has  shown  that  prolonged  and  intimate 
association  with  a  leper  does  not  as  a  rule  result  in  infection. 

Leprosy  of  Rats. — Stefansky1  has  reported  a  disease  of  rats  which 
resembles  human  leprosy  in  a  striking  manner;  like  the  disease  in 
man  the  lesions,  which  consist  essentially  of  glandular  enlargement, 
subcutaneous  infiltration  and  induration,  alopecia,  and  frequently 
deep-seated  cutaneous  ulcerations,  contain  large  numbers  of  acid- 
fast  bacilli  which  resemble  Bacillus  leprse  both  morphologically  and 
in  their  collection  in  large  numbers  in  the  localized  swellings.  Dean2 
has  corroborated  the  observation  and  also  found  acid-fast  bacilli  in  the 
nasal  secretion  of  the  rats.  He  also  isolated  a  diphtheroid  bacillus 
similar  to  that  of  Kedrowski.  The  disease  is  wide-spread  among 
rats,  being  reported  in  Russia,3  Berlin,4  Australia,5  and  the  United 
States.6  It  is  found  in  areas  free  from  human  cases,  as  human  leprosy 
is  found  in  locations  where  the  disease  is  not  found  in  rodents. 
Whether  the  human  and  rat  leprosy  bacilli  are  identical  or  not  is  not 
finally  decided;  Mezinescu7  and  Schmitt8  have  shown  by  the  method 
of  complement  fixation  that  the  sera  of  human  and  rat  lepers  mutually 
exhibit  complete  reactions. 

ACID-FAST   BACILLI   OTHER   THAN   BACILLUS   TUBERCULOSIS 
AND   BACILLUS   LEPR^I. 

Following  the  discovery  of  the  tubercle  and  leprosy  bacilli,  which 
exhibit  the  striking  phenomenon  of  "acid-fastness,"  a  number  of 
bacteria  presenting  the  same  general  staining  reactions  have  been 
described.  They  are  somewhat  widely  distributed  in  nature,  being 

1  Centralbl.  f.  Bakt.,  Orig.,  1903,  xxxiii,  481. 

2  Ibid.,  xxxiv,  222. 

3  Stefansky,  loc.  cit. 

4  Rabinovitsch,  Centralbl.  f.  Bakt.,  Orig.,  1903,  xxxiii,  577. 
8  Bull.  Jour,  of  Australasia,  1907,  263. 

6  Wherry,    Jour.    Am.    Med.   Assn.,    1908,   1,   1903.     McCoy,    Public    Health  Rep., 
July  10,  1908. 

7  Compt.  rend.  Soc.  biol..  1908,  Ixiv,  514;    1909,  Ixvi,  56. 

8  University  of  California  Pub.  in  Path.,  1911,  ii,  29. 


ACID-FAST  BACILLI  OTHER  THAN  BACILLUS  TUBERCULOSIS    469 

occasionally  isolated  from  water,  grass  or  manure,  and  they  are  also 
found  in  association  with  man  and  the  higher  domestic  animals, 
frequently  occurring  as  parasites  'upon  the  skin,  less  commonly  in 
the  nasal  secretion  or  sputum. 

Morphologically  the  acid-fast  bacteria  of  the  non-pathogenic  type 
are  somewhat  shorter  and  relatively  thicker  than  the  tubercle  bacillus 
(human  type),  and  in  culture  they  not  infrequently  grow  in  filaments 
and  exhibit  branching.  Upon  artificial  media,  furthermore,  develop- 
ment is  relatively  rapid;  growth  usually  appears  within  forty-eight 
hours,  even  at  25°  to  27°  C.  The  usual  type  of  growth  is  an  irregular, 
wrinkled  layer,  waxy  in  appearance  and  of  variable  color  from  gray 
to  yellow,  orange  or  even  brown.1  The  injection  of  considerable 
amounts  of  the  bacteria  into  guinea-pigs  may  lead  to  the  formation 
of  granulation  tissue  nodules,  for  the  organisms  are  very  insoluble  in 
the  body  juices;  superficially  these  nodules  may  resemble  tubercles, 
but  they  differ  in  two  important  particulars — they  df>  n*t  develop 
progressively  but  are  limited  to  the  site  of  inoculation,  and  they  tend 
to  soften  gradually  and  eventually  to  suppurate  and  heal  spon- 
taneously with  scar  tissue  formation. 

The  best-known  members  of  the  group  are:  Bacillus  phlei,  includ- 
ing the  various  bacilli  isolated  from  grasses  and  manure;  the  smegma 
bacillus,  which  grows  on  the  genitalia  and  the  cerumen;  and  the 
nasal  secretion  type  found  occasionally  on  the  skin,  in  the  nasal  secre- 
tion, the  sputum,  tonsillar  exudates  and  rarely  in  gangrene  of  the 
lungs.  It  is  very  probable  that  the  tubercle  bacilli  of  cold-blooded 
animals — (ichthic  tubercle  bacilli) — fish,  turtles,  snakes,  and  the 
"  Blindschleiche"  bacillus  belong  to  this  group. 

The  Smegma  Bacillus. — Alvarez  and  Tarbel2  found  an  organism 
on  the  external  genitalia  and  around  the  anus  which  is  very  similar 
morphologically  and  in  staining  reaction  to  the  tubercle  bacilms. 
Moeller3  and  others  have  confirmed  this  observation.  The  organism 
was  called  the  smegma  bacillus.  It  has  been  regarded  by  many  as 
identical  with  a  bacillus  described  in  1884  by  Lustgarten  as  the 
causative  organism  of  syphilis. 

The  cultivation  of  both  of  these  organisms  in  artificial  media  is 
difficult,  and  it  is  not  definitely  proven  that  it  has  been  accomplished. 

The  practical  importance  of  these  organisms  lies  in  the  fact  that 

1  Tuberculin  is  not  produced  in  cultures  in  artificial  media. 

2  Arch.  d.  phys.  norm,  et  path.,  1885,  No.  7. 

3  Centralbl.  f.  Bakt,,  Orig.,  1902,  xxxi,  278. 


470  LEPROSY  AND  ACID-FAST  BACTERIA 

they  may  be  confused  with  the  tubercle  bacillus  in  the  examination 
of  urine  or  feces  for  the  latter.  The  organisms  are  not  pathogenic 
for  guinea-pigs  and  a  distinction  between  the  smegma  bacillus  and 
the  tubercle  bacillus  may  be  effected  in  this  way. 

The  Nasal  Secretion  Bacillus. — Karlinski1  isolated  an  organism 
from  the  nasal  secretion  of  a  man  which  possessed  morphological  and 
staining  peculiarities  very  similar  to  those  of  the  tubercle  bacillus. 
Similar  or  identical  organisms  have  been  isolated  from  tonsillar 
exudates,  from  a  few  cases  of  pulmonary  gangrene  and  from  sputum. 

The  organism  grows  readily  on  ordinary  media.  It  presents  no 
definite  peculiarities  of  staining  which  would  distinguish  it  from  the 
tubercle  bacillus,  and  its  occasional  occurrence  in  the  nasal  and  oral 
secretions  necessitates  great  care  in  distinguishing  it  from  that  organism. . 

The  organism  is  non-pathogenic  for  guinea-pigs  and  in  suspicious 
cases  a  differentiation  between  the  nasal  secretion  bacillus  and  the 
tubercle  bacillus  can  be  made  through  this  animal. 

.Bacillus  Phlei. — Synonyms. — Grass  bacillus,  Timothy  grass  bacillus, 
Mist  bacillus. 

Historical. — The  most  important  investigations  of  the  saprophytic 
acid-fast  bacilli  are  those  of  Moeller.2  The  members  of  this  group, 
designated  as  Grass  bacillus  I  and  II,  from  hay  infusions,  and  the 
Mist  bacillus  from  manure,  are  very  similar  in  their  general  staining 
and  cultural  reactions — so  similar  that  the  slight  differences  noticed 
are  of  insufficient  magnitude  to  warrant  their  separation  into  distinct 
types.  For  the  present  they  are  best  regarded  as  variants  of  the 
same  organism. 

Morphology. — Bacillus  phlei  resembles  the  tubercle  bacillus  (human 
type)  in  its  morphological  characters,  except  that  it  is  somewhat 
shorter  and  relatively  thicker.  Occasionally  isolated  organiHife  exhibit 
swollen,  club-shaped  ends,  and  branching  is  frequently  observed  in 
cultures  in  artificial  media.  They  stain  with  difficulty  and  resist  the 
combined  decolorizing  action  of  mineral  acids  and  alcohol. 

Isolation  and  Culture. — The  organisms  grow  readily  and  rapidly  on 
ordinary  media,  and  after  three  or  four  days'  incubation,  the  colonies 
are  round,  somewhat  waxy  in  appearance,  and  vary  in  diameter  from 
2  to  5  mm.  Typically  colonies  are  yellowish  to  a  dark  orange  in  color. 
Subcultures  are  obtained  very  readily. 

1  Centralbl.  f.  Bakt.,  1901,  xxxix,  525. 

2  Deutsch.  med.  Wchnschr.,  1898,  No.  24;    Centralbl.  f.  Bakt.,  1899,  xxv,  369;    1901, 

xxx,  sis. 


ACID-FAST  &ACILLI  OTHER  THAN  BACILLUS  TUBERCULOSIS   471 

Pathogenesis. — Bacillus  phlei  is  not  pathogenic  for  man,  so  far  as 
is  known,  but  the  introduction  of  large  numbers  of  the  organisms 
into  the  peritoneal  cavity  of  guinea-pigs  leads  to  the  formation  of 
localized  nodules  which  eventually  soften  and  contain  a  purulent, 
somewhat  caseous  mass.  Typical  tubercles  with  giant-cell  and  epithe- 
lioid-cell  formation  are  not  observed.  Moderate  doses  do  not  cause 
death,  but  very  large  doses  frequently  lead  to  fatal  results.  The 
inoculated  animals  fail  to  give  any  reaction  whatsoever  with  tuberculin 
derived  from  human  or  bovine  cultures. 

The  organisms  are  of  practical  importance  because  they  may  be 
confused  with  the  tubercle  bacillus.  A  simple  microscopic  examina- 
tion may  in  rare  instances  lead  to  error,  but  the  correct  differentiation 
between  these  organisms  and  the  tubercle  bacillus  may  be  safely 
arrived  at  by  their  injection  into  guinea-pigs  and  the  subsequent 
negative  reaction  with  a  fairly  large  dose  of  tuberculin. 

The  Butter  Bacillus. — This  organism  was  first  described  by 
Rabinovitsch,1  and  subsequently  her  observations  were  confirmed  and 
extended  by  Petri.2 

Morphologically  the  organisms  are  very  similar  to  tubercle  bacilli, 
but  they  are  relatively  less  acid-fast.  Differentiation  between  the 
butter  bacillus  and  the  tubercle  bacillus,  however,  can  not  be  made 
upon  this  basis.  The  organisms  grow  in  culture  media  very  like  the 
grass  bacilli.  In  broth  the  medium  remains  clear  and  the  organisms 
form  a  thick,  wrinkled  pellicle  on  the  surface.  Very  frequently  there 
is  a  distinct  ammoniacal  odor  to  the  broth,  and  it  is  said  that  they 
form  small  amounts  of  indol. 

So  far  as  is  known  the  butter  bacilli  are  non-pathogenic  for  man 
and  the  lesions  they  induce  in  guinea-pigs  are  very  similar  to  those 
produced  by  the  grass  bacilli.  They  are  chiefly  confusing  when  they 
are  found  in  milk  and  butter  because  of  their  resemblance  to  the  bovine 
tubercle  bacillus.  A  distinction  between  the  butter  bacillus  and  the 
bovine  tubercle  bacillus  can  be  definitely  made  by  injection  into  guinea- 
pigs.  The  lesions  are  not  tubercular  in  nature,  and  the  animals  fail 
to  react  to  tuberculin. 

1  Ztschr.  f.  Hyg.,  1897,  xxvi,  90.  2  Hyg.  Rund.,  August  15,  1897. 


CHAPTER  XXV. 
ANAEROBIC  BACTERIA. 

BACILLUS   TETANI. 

THE  infectious  nature  of  tetanus  was  first  clearly  demonstrated  by 
inoculating  rabbits  subcutaneously  with  pus  from  a  human  case  of 
the  disease.  This  experiment,  which  reproduced  the  essential  clinical 
features  of  the  disease  and  killed  the  animals,  was  performed  by  Carle 
and  Rattoni1  in  1884.  The  same  year  Nicolaier2  saw  the  tetanus 
bacillus  in  laboratory  animals  which  were  inoculated  subcutaneously 
with  garden  soil,  at  the  site  of  injection.  It  remained  for  Kitasato,3 
however,  to  grow  the  tetanus  bacillus  in  pure  culture  and  to  definitely 
transmit  the  disease  to  laboratory  animals  through  pure  cultures  of 
the  organism. 

Morphology. — Bacillus  tetani  is  a  long,  slender  bacillus  with  rounded 
ends,  measuring  from  0.3  to  0.8  micron  in  diameter  and  from  2  to  5 
microns  in  length,  which  commonly  occurs  singly  and  in  pairs  in  young 
cultures;  in  older  cultures  the  organisms  tend  to  form  long  chains. 
It  tends  to  degenerate  in  older  cultures,  leaving  free  spores  and  involu- 
tion forms.  The  bacillus  is  slightly  motile  in  recently  inoculated 
cultures  and  possesses  from  sixty  to  eighty  peritrichic  flagella.4  Cap- 
sules are  not  produced  by  Bacillus  tetani.  It  stains  readily  with 
ordinary  dyes  and  is  Gram-positive.  Spores  are  readily  formed  under 
anaerobic  conditions,  which  are  so  characteristic  in  appearance  and 
constant  in  occurrence  that  they  are  of  diagnostic  importance.  The 
spores  are  spherical,  greater  in  diameter  than  the  bacillus  (measuring 
1  to  1.5  microns  in  diameter)  and  occur  at  one  end  of  the  rod,  giving 
it  the  appearance  of  a  drumstick  or  plectridium.  The  rate  of  spore 
formation  in  artificial  media  appears  to  be  greatly  influenced  by  the 
temperature  of  incubation:  at  20°  C.  spores  appear  in  from  seven  to 
eight  days;  at  37°  C.  they  are  usually  found  in  large  numbers  after 
one  to  two  days;  at  43°  C.  the  organisms  grow  slowly  and  form  but 
few  spores;  but  little  toxin  is  produced  at  this  temperature. 

1  Giornale  della  R.  accad.  di  Med.  di  Torino. 

2  Deutsch.  med.  Wchnschr.,  «84,  No.  52;    Inaug.  Diss.,  Gottingen,  1885. 

3  Deutsch.  med.  Wchnschr.,  ]»,  No.  31;    Ztschr.  f.  Hyg.,  1889,  vii,  225. 
4Schwarz,  Lo  sperimentale,  iM,  p.  373.     Grandi,  Centralbl.  f.  Bakt.,  Orig.,  1903, 

xxxiv,  97. 


BACILLUS  TETAN1  473 

Isolation  and  Culture. — Pure  cultures  of  tetanus  bacilli  are  difficult 
to  obtain  from  the  soil  or  from  other  sources,  where  it  exists  in  asso- 
ciation with  other  bacteria.  Kitasato1  succeeded  in  isolating  pure 
cultures  by  alternately  incubating  alkaline  broth  containing  tetanus 
and  other  bacteria  anaerobically  at  37°  C.  for  forty-eight  hours,  then 
heating  the  culture  to  80°  C.  for  thirty  minutes  to  destroy  non-spore- 
forming  organisms.  Theobald  Smith2  has  devised  a  method  for  obtain- 
ing pure  cultures  of  spore-forming  anaerobes,  including  tetanus  bacilli, 
which  is  far  more  successful  in  practice  than  the  Kitasato  method. 
Fermentation  tubes  containing  sugar-free  broth  slightly  alkaline  in 
reaction  and  bits  of  sterile  tissue  (kidney  or  liver  from  rabbits  or 


FIG.  65. — Bacillus  tetani,  spore  formation.      X  1000.     (Giinther.) 

guinea-pigs)  are  inoculated  with  the  suspected  material  and  incubated 
at  37°  C.  for  forty-eight  hours.  The  growth  of  anaerobic  organisms 
is  much  more  luxuriant  in  this  tissue  medium  than  in  similar  media 
without  the  tissue.3  Tetanus  spores  are  formed  abundantly  around 
the  bit  of  tissue,  and  after  forty-eight  hours  of  incubation  the  culture 
is  again  heated  to  80°  C.  for  thirty  minutes  to  kill  non-spore-forming 
organisms.  The  spore-containing  medium  which  collects  at  the  lowest 
part  of  the  fermentation  tube  around  the  bit  of  tissue  is  reinoculated 
into  a  fresh  fermentation  tube  of  the  same  medium,  and  the  process 
is  repeated  in  detail.  It  is  advisable  to  use  a  pipette  to  remove  the 
material  for  inoculation  in  order  to  insure  an  abundance  of  spores. 
The  success  of  the  procedure  is  readily  controlled  by  stained  prepara- 

1  Loc.  cit. 

2  Jour.  Boston  Soc.  Med.  Sci.,  1899,  340;   Jour.  Med.  Research,  1905,  xiv,  193. 

3  This  method  has  been  rediscovered  by  TizzonT  (Centralbl.  f.  Bakt.,  Orig.,  1905, 
xxxiv,  619),  and  others. 


474  ANAEROBIC  BACTERIA 

tions  made  from  the  material  inoculated  each  time,  and  the  process 
is  repeated  until  microscopical  examination  reveals  a  sufficient  number 
of  bacilli  of  characteristic  appearance.  Finally,  the  enriched  culture, 
after  a  final  heating  to  80°  C.  for  thirty  minutes,  is  plated  out  anaerob- 
ically  upon  blood  agar  plates. 

Tetanus  bacilli  characteristically  produce  a  wide  zone  of  hemolysis 
around  the  colonies  on  blood  agar,  and  the  colonies  themselves  tend 
to  spread  rapidly.1 

Growth  in  Media. — The  tetanus  bacillus  is  typically  an  obligate 
anaerobe,  although  various  successful  attempts  to  induce  aerobic 
development  have  been  recorded.2  The  characteristic  reactions  and 
products  of  the  organism,  however,  are  detected  only  in  anaerobic 
cultures.  On  anaerobic  agar  plates  the  colonies  are  filamentous ; 
under  the  lower  powers  of  the  microscope  they  resemble  densely  matted 
strands  of  cotton  fiber.  Gelatin  colonies  are  quite  similar,  except 
that  in  sugar-free  gelatin  liquefaction  takes  place  after  three  to  five 
days'  incubation.  The  growth  in  deep  stab  cultures  is  distinctive; 
the  organisms  grow  away  from  the  line  of  inoculation  at  right  angles, 
producing  an  appearance  which  has  been  likened  to  an  inverted  pine 
tree.  The  growth  fails  to  reach  the  surface  of  the  medium,  however, 
indicating  the  anaerobic  nature  of  the  bacteria.  Milk  appears  to  be 
a  favorable  medium  for  their  development.  A  slight  acidity  is  pro- 
duced, but  no  coagulation  or  peptonization.  Slightly  alkaline,  sugar- 
free  broth  overlaid  with  a  layer  of  paraffin  or  paraffin  oil3  is  a  favorable 
medium;  the  organisms  produce  a  well-defined  turbidity  after  twenty- 
four  to  forty-eight  hours'  incubation  at  37°  C.  which  increases  in 
intensity  for  about  fourteen  days,  at  the  end  of  which  time  the  growth 
begins  to  settle  to  the  bottom  of  the  flask.  Cultures  of  tetanus  bacilli 
usually  possess  a  very  disagreeable  odor. 

Conditions -of  Growth. — Bacillus  tetani  is  an  obligate  anaerobe,  but 
strains  may  be  gradually  accustomed  to  oxygen  so  that  eventually 
they  will  grow  slowly  even  in  the  presence  of  air.  They  lose  their 
toxin-producing  powers,  however,  under  these  conditions.4  The 

1  Boulton  and  Fisch,  Trans.  Am.  Phys.,   1902,  463.     It  is  occasionally  possible  to 
isolate  tetanus  bacilli  directly  from  mixtures  by  inoculating  dextrose  agar  with  dilute 
suspensions  of  the  suspected  material,  which  has  previously  been  heated  to  80°  C.  for 
thirty  minutes.     The  agar  is  drawn  up  into  long,  sterile  glass  tubes  of   approximately 
5  mm.  bore,  and  the  ends  sealed  by  heating.     After  forty-eight  hours'  incubation  at 
37°  C.  characteristic  colonies  are  visible  through  the  glass.     The  outside  of  the  tube 
is  carefully  sterilized  and  cut  with  a  file  close  to  the  desired  organisms,  which  may  be 
removed  by  a  sterile  capillary  pipette. 

2  Ferran,  Centralbl.  f.  Bakt.,  1898,  xxiv,  28. 

3  Park,  Centralb.  f.  Bakt.,  1901,  xxix,  445.  4  Ferran,  loc.  cit. 


BACILLUS  TETANI  475 

organisms  grow  well  in  a  vacuum  or  in  an  atmosphere  of  hydrogen  or 
nitrogen;  they  grow  poorly  or  not  at  all  in  an  atmosphere  of  carbon 
dioxide.  Growth  does  not  take  place  below  14°  C.  or  above  45°  C.; 
the  optimum  is  37°  C.  Growth  is  slow  at  20°  C.  and  spore  formation 
proceeds  sluggishly.  At  37°  C.  growth  and  spore  formation  are  opti- 
mum. Growth  is  fairly  rapid  at  43°  C.,  but  spore  formation  is  greatly 
interfered  with,  and  above  45°  C.  growth  ceases. 

The  spores  are  very  resistant  to  drying;  when  kept  in  the  dark  and 
cool  they  may  survive  for  years.  Henri  jean1  has  found  that  tetanus 
spores  may  remain  viable  and  virulent  for  nearly  eleven  years.  The 
resistance  of  spores  to  heat  is  a  subject  on  which  there  is  great  difference 
of  opinion.  Theobald  Smith2  has  studied  the  resistance  of  tetanus 
spores  under  varying  conditions,  and  his  results  are  the  most  trust- 
worthy available.  In  gelatin  sporulation  is  relatively  feeble  and 
spores  formed  in  this  medium  do  not  appear  to  be  very  resistant. 
He  states  that  a  majority  of  tetanus  spores  survive  an  exposure  to 
flowing  steam  for  forty  minutes,  occasionally  for  sixty  minutes;  and 
in  one  experiment  a  seventy-minute  exposure  did  not  destroy  all 
spores.  Morax  and  Marie3  have  found  that  dried  spores  are  killed 
by  an  exposure  to  dry  heat  at  125°  C.  for  twenty  minutes.  A  5  per 
cent,  solution  of  carbolic  acid  kills  tetanus  spores  in  about  ten  hours; 
mercuric  chloride  in  a  dilution  of  1  to  1000  kills  them  in  three  hours; 
the  addition  of  0.5  per  cent,  hydrochloric  acid  increases  the  germicidal 
action  of  both  carbolic  acid  and  mercuric  chloride.  A  1  per  cent, 
solution  of  silver  nitrate  kills  tetanus  spores  in  one  minute,  and  a 
0.1  per  cent,  solution  in  five  minutes.  lodoform  is  said  to  be  par- 
ticularly efficient. 

Products  of  Growth. — Among  the  products  of  metabolism  of  the 
tetanus  bacillus  in  sugar-free  media  are  indol,  hydrogen  sulphide,  and 
mercaptan,  which  impart  an  extremely  disagreeable  odor  to  cultures 
of  the  organism.  Bacillus  tetani  ferments  dextrose  and  maltose,  pro- 
ducing acid,  partly  lactic,  as  well  as  considerable  amounts  of  carbon 
dioxide  and  hydrogen.  Bioses  other  than  maltose,  and  polysaccharides 
are  not  fermented. 

The  most  characteristic  and  striking  metabolic  product,  however,  is 
an  extremely  potent,  soluble,  extracellular  toxin.  This  toxin,  as  Ehrlich 
has  shown,4  contains  at  least  two  distinct  components  in  varying 

1  Ann.  de  la  Soc.  m6d.-chir.  de  Li&ge,  1891,  367. 

2  Jour.  Am.  Med.  Assn.,  1908,  1,  929. 

3  Ann.  Inst.  Past.,  1902,  421. 

4  Berl.  klin.  Wchnschr.,  1898,  No.  12. 


476  ANAEROBIC  BACTERIA 

proportions,  which  may  be  recognized  by  their  respective  physio- 
logical actions:  tetanospasmin,  a  neurotoxin,  which  is  relatively 
thermostable  and  produces  the  characteristic  tonic  contractions  or 
spasms  which  characterize  the  disease  tetanus;  and  tetanolysin,  a 
relatively  thermolabile  hemo toxin  which  dissolves  red  blood  cells.  It 
is  doubtful  if  the  tetanolysin  is  ordinarily  of  clinical  importance. 

Tetanus  toxin  appears  to  be  produced  only  in  sugar-free  media 
under  anaerobic  conditions.  Buchner1  seems  to  have  detected 
small  amounts  of  true  tetanus  toxin  in  cultures  of  tetanus  bacilli 
grown  in  a  modified  Uschinsky  medium  containing  asparagin  and 
certain  inorganic  salts.  Brieger,  on  the  contrary,2  maintains  that  the 
toxin  is  produced  only  when  the  organisms  are  grown  in  albuminous 
media. 

Tetanus  toxin  is  best  prepared  in  slightly  alkaline  peptone-meat 
infusion  broth  containing  0.1  per  cent,  of  dextrose.  The  dextrose  is 
added  to  insure  a  large  initial  development  of  bacteria  which  as  soon 
as  the  sugar  is  exhausted  (within  twenty-four  hours)  attacks  the  pro- 
tein constituents  of  the  medium,  forming  from  them  the  tetanus 
toxin.3  It  is  essential  to  heat  the  medium  to  the  boiling  point  and 
cool  it  rapidly  immediately  before  inoculation  to  drive  out  all  traces 
of  oxygen.  Anaerobic  conditions  are  most  easily  obtained  and  main- 
tained by  overlaying  the  broth  with  pure  paraffin  oil,  according  to  the 
method  of  Park.4  Incubation  should  be  maintained  at  37°  C.  for  seven 
to  ten  days.  The  toxin  appears  to  lose  somewhat  in  potency  if  incu- 
bated for  a  longer  period.  The  potency  of  the  toxin  prepared  in  this 
manner  varies  considerably,  being  influenced  by  the  composition  and 
reaction  of  the  medium  and  the  degree  of  anaerobiosis.  Tetanus 
bacilli  retain  their  ability  to  produce  toxin  with  great  tenacity  and 
regularity,  even  after  prolonged  artificial  cultivation.  At  the  end  of 
the  period  of  incubation  the  broth  is  rapidly  filtered  through  sterile 
unglazed  porcelain  filters  into  dark-colored  bottles,  which  are  com- 
pletely filled  to  exclude  oxygen  after  the  addition  of  0.5  per  cent, 
carbolic  acid.  It  should  be  kept  in  a  cool,  dark  place  under  anaerobic 
conditions.  A  small  amount  of  the  broth  containing  toxin  thus 
obtained,  freed  from  bacteria,  will  liquefy  gelatin,  thus  showing  that 
a  peptonizing  ferment  is  present  in  the  filtrate,  either  inherent  in  the 
toxin  or  in  association  with  it.  According  to  Fermi  and  Pernossi,f 

1  Munchen.  med.  Wchnschr.,  1893,  No.  24,  450. 

2  Ztschr.  f.  Hyg.,  1895,  xix,  102. 

3  Kendall,  Boston  Med.  and  Surg.  Jour.,  1913,  clxviii,  825. 

4  Loc.  cit.  6  Centralbl.  f.  Bakt.,  1894,  xv,  303. 


BACILLUS  TETANI  477 

the  gelatin-liquefying  ferment  (peptonizing  ferment)  has  nothing  to 
do  with  the  toxin;  it  is  quite  distinct  from  it. 

Properties  of  Tetanus  Toxin. — Tetanus  toxin  is  unstable.  Exposure 
of  broth  filtrates  containing  tetanus  toxin  to  55°  C.  for  an  hour  and 
a  half,  twenty  minutes  at  60°  C.,  or  five  minutes  at  65°  C.,  reduces 
the  potency  to  a  very  considerable  degree.1  For  the  complete  destruc- 
tion of  the  toxin,  however,  considerable  heating  is  necessary.  The 
toxin  is  particularly  susceptible  to  light.  According  to  Fermi  and 
Pernossi,2  fifteen  to  eighteen  hours'  exposure  to  daylight  destroys  it.3 
Tetanus  toxin  is  destroyed  by  gastric  and  by  tryptic  digestion.4  Dried 
tetanus  toxin  is  more  stable  to  physical  agents  and  to  heat  than  toxin 
in  solution.  Morax  and  Marie5  have  shown  that  dried  tetanus  toxin  is 
not  destroyed  by  an  exposure  to  dry  heat  of  120°  C.  for  fifteen  minutes. 

Purification  of  Toxin. — Tetanus  toxin  may  be  obtained  in  a  partially 
purified  state  by  precipitating  the  broth  in  which  it  is  contained 
with  saturated  ammonium  sulphate,  dialyzing  the  salts  from  the 
precipitate  and  drying  the  salt-free  residue  in  vacuo.6  The  dried  toxin, 
if  kept  in  a  cool,  dark  place,  remains  potent  for  many  months. 

Tetanus  toxin  is  one  of  the  most  potent  known:  as  little  as  0.0001 
c.c.  of  the  toxic  broth  frequently  kills  a  15-gram  mouse.  Purified 
toxin,  prepared  by  precipitation  with  ammonium  sulphate,  will  kill 
a  mouse  of  the  same  weight  if  but  0.00005  gram  is  injected.  Man  and 
the  horse  are  very  susceptible  to  the  tetanus  toxin.  Knorr7  estimated 
that  a  gram  of  horse  was  twelve  times  as  susceptible  to  the  tetanus 
toxin  as  a  gram  of  mouse,  and  three  hundred  times  as  susceptible  as 
a  gram  of  hen.  The  reptilia  are  practically  non-susceptible:  toxin 
injected  into  these  animals  circulates  in  the  blood  stream  without 
causing  symptoms  and  it  is  finally  eliminated. 

Action  of  Tetanus  Toxin. — Even  when  massive  doses  of  toxin  are 
injected  into  susceptible  animals,  a  latent  period  exists  between  the 
time  of  inoculation  and  the  appearance  of  symptoms,  which  can  not 
be  reduced  below  eight  hours.8  The  incubation  period  increases  when 


1  Kitasato,  Ztschr.  f.  Hyg.,  1891,  x,  267.  2  Ztschr.  f.  Hyg.,  1894,  xvi,  385. 

3  For  a  full  discussion  of  the  physical  properties  of  tetanus  toxin  see  Fermi  and  Per- 
nossi, Centralbl.  f.  Bakt.,  1894,  xv,  303. 

4  Baldwin  and  Levene,  Jour.  Med.  Research,  1901,  vi,  120. 

5  Ann.  Inst.  Past.,  1902,  419-420.         6  Brieger  and  Cohen,  Ztschr.  f.  Hyg.,  1893,  xv,  8. 

7  Munchen.  med.  Wchnschr.,  1898,  321. 

8  Courmont  and  Doyen,  Arch,  de  Phys.,  1893;  Goldschneider  and  Flatau  (Kong.  f. 
inn.  Med.,  Berlin,  June  11,  1897;     Deutsch.  med.  Wchnschr.,  1897,  Vereinsbl.  No.  18, 
129;    Fort.  d.  Med.,  1897,  609)   have  noticed  however,  that  changes  in  the  anterior 
horn  ganglion  cells  of  the  spinal  cords  of  rabbits  are  demonstrable  two  hours  after 
injection  of  tetanus  toxin. 


478  ANAEROBIC  BACTERIA 

smaller  amounts  of  toxin  are  used;  symptoms  may  not  appear  until 
two  or  three  days,  or  even  a  week  after  inoculation.  Subfatal  doses 
of  tetanus  toxin  administered  to  experimental  animals  give  rise  to 
local  symptoms  which  are  frequently  the  only  signs  observed.  The 
incubation  period  of  the  natural  infection  in  man  is  usually  about 
fourteen  to  sixteen  days.  It  may  be  stated  as  a  general  rule  that  the 
shorter  the  incubation  period,  the  higher  the  mortality.  The  "site  of 
inoculation  of  the  tetanus  toxin  influences  the  character  of  the  symp- 
toms and  the  incubation  period  quite  materially.  Subcutaneous 
injections  are  usually  followed  by  symptoms  (spasms)  which  affect 
the  muscles  nearest  the  site  of  inoculation  as  a  rule.  Intravenous 
injections  usually  cause  a  generalized  spasm.1  When  toxin  is  intro- 
duced directly  into  the  central  nervous  system  smaller  doses  cause 
death  and  the  symptoms  develop  much  more  rapidly.  There  is  great 
restlessness  in  these  cases  before  the  characteristic  spasms  occur,  and 
the  spasms  are  epileptiform  in  character.  The  toxin  is  supposed  to 
exert  a  harmful  effect  on  the  central  nervous  system,  which  it  reaches 
by  way  of  the  nerve  trunks.  Donitz,2  and  Wassermann  and  Takaki3 
have  shown  that  mixtures  of  brain  tissue  (especially  the  gray  substance) 
and  tetanus  toxin  are  practically  without  effect  when  they  are  injected 
into  susceptible  animals,  indicating  that  a  firm  union  has  taken  place 
between  the  tissue  and  the  toxin.  This  union  will  take  place  in  vitro. 
The  spleen,  liver,  kidney  and  other  non-nerve-containing  tissue  have 
little  or  no  neutralizing  power  for  tetanus  toxin.  Metchnikoff4  and 
Blumenthal5  have  determined  experimentally  that  the  brain  tissue 
of  pigeons  and  hens,  which  are  almost  refractory  to  tetanus  toxin, 
possess  but  little  neutralizing  power  for  it.6  Asakawa7  has  corroborated 
these  results  and  has  also  shown  that  the  toxin  may  circulate  for  some 

1  Ransom,  Deutsch.  med.  Wchnschr.,   1893.     Marie  and  Morax,  Ann.   Inst.  Past. 

1902,  xvi,  818. 

2  Deutsch.  med.  Wchnschr.,  1897,  248. 
s  Berl.  klin.  Wchnschr.,  1898,  xxxv,  5. 

4  Ann.  Inst.  Past.,  1898,  81. 

5  Deutsch.  med.  Wchnschr.,  1898. 

6  There  appears  to  be  some  combining  power  of  the  brain  tissue  of  non-susceptible 
animals,  as  hens  and  pigeons,  for  tetanus  toxin,  however.     A  possible  explanation  for 
this  phenomenon  is  furnished  by  Landsteiner  and  Von  Eisler  (Centralbl.  f.  Bakt.,  Orig., 

1903,  xxxiv,  567;  1905,  xxxix,  315).      They  found   that  lipoids  would  combine  with 
tetanus  toxin  at  least  to  a  limited  degree.     Levene  (Biochem.  Ztschr.,  1911,  xxxiii,  225; 
xxxiv,  495)  has  shown  that  tetanus  toxin  will  unite  not  only  with  lipoids  but  with  fats 
and  similar  substances.     Marie  and  Tiffeneau  (Ann.  Inst.  Past.,  1908,  xxii,  289,  644) 
have  discovered  that  although  a  small  amount  of  tetanus  toxin  may  be  bound  by  lipoidal 
substances  in  the  brain  in  susceptible  animals,  the  greater  part  of  it  is  bound  by  albumi- 
nous substances.     They  believe  .that  the  essential   albuminous  substances  necessary 
for  this  union  are  absent  or  inactive  in  non-susceptible  animals. 

7  Centralbl.  f.  Bakt.,  1898,  xxiv,  166,  234. 


BACILLUS  TETANI  479 

time  in  the  blood  of  these  animals  before  it  is  excreted.  Donitz1 
and  Knorr2  have  shown  that  tetanus  toxin  disappears  rather  rapidly 
from  the  blood  stream  of  susceptible  animals,  on  the  contrary,  and 
almost  coincidently  with  its  disappearance  the  symptoms  become 
manifest.  Wolff3  states  that  the  injection  of  tetanus  toxin  into  experi- 
mental animals  in  small  doses  produces  a  lymphocytosis. 

How  Tetanus  Toxin  is  Absorbed. — The  brilliant  researches  of  Meyer 
and  Ransom4  have  shown  that  tetanus  toxin  is  absorbed  by  the  per- 
ipheral nerve  end-organs  and  travels  along  the  axis  cylinders  of  the 
nerves  to  the  central  nervous  system.  The  spasms,  which  are 
characteristic  of  tetanus,  are  supposed  to  be  of  central  origin,  and  the 
experiments  of  Gumprecht5  would  suggest  that  this  is  the  case.  He 
cut  the  motor  nerves  to  a  limb  and  thus  prevented  the  tonic  contrac- 
tions in  that  part.  Zupnik6  believes  that  the  spasms  may  be  either  of 
peripheral  or  central  origin,  the  symptoms  elicited  depending  largely 
upon  the  reflex  irritability  of  the  medulla  or  cord.  This  view  has  not 
been  substantiated. 

Tetanus  Antitoxin. — The  injection  of  tetanus  toxin  in  very  small, 
sub-fatal  doses,  which  are  gradually  increased,  or  of  toxin  weakened 
by  chemicals,  as  iodine  trichloride,  induces  immunity  in  horses  or 
other  susceptible  animals,  which  is  manifested  by  the  gradual  appear- 
ance of  a  specific  antitoxin  in  the  blood.  This  antitoxin  will  neutralize 
tetanus  toxin  both  in  vitro  and  in  vivo;  it  will  prevent  the  development 
of  tetanus  in  experimental  animals,  provided  it  is  given  before  or 
immediately  following  the  injection  of  toxin.  Donitz7  has  shown 
that  as  many  as  twelve  fatal  doses  of  toxin  may  be  neutralized  -jby  1 
c.c.  of  a  0.001  to  0.002  dilution  of  antitoxin,  provided  the  toxin  and 
antitoxin  are  mixed  before  injection.  Four  minutes  after  injection 
of  1  c.c.  of  toxin,  1  c.c.  of  1  to  600  dilution  of  antitoxin  is  required  for 
neutralization;  eight  minutes  after  injection  of  the  same  amount  of 
toxin,  1  c.c.  of  1  to  200  dilution  of  antitoxin  is  required  to  protect  the 
animal,  and  fifteen  minutes  after  the  injection  of  1  c.c.  of  toxin,  1 
c.c.  of  1  to  100  dilution  of  antitoxin  is  required.  These  experiments 
illustrate  clearly  the  necessity  of  administering  tetanus  antitoxin  at 
the  earliest  possible  moment  to  obtain  favorable  results. 

Inasmuch  as  the  toxin  appears  to  reach  the  central  nervous  system 

1  Deutsch.  med.  Wchnschr.,  1897,  No.  27. 

2  Miinchen.  med.  Wchnschr.,  1898,  Nos.  11  and  12. 

3  Berl.  klin.  Wchnschr.,  1904,  xli,  1273. 

4  Arch.  f.  exp.  Pharm.  u.  Path.,  1903,  xlix,  369.  5  Pfliiger's  Archiv,  1895. 

6  Deutsch.  med.  Wchnschr.,  1900,  837.  7  Ritchie,  Jour,  of  Hyg.,  ii. 


480  ANAEROBIC  BACTERIA 

by  way  of  the  nerves,  while  the  antitoxin  circulates  in  the  blood  stream, 
it  is  not  surprising,  as  Welch1  has  pointed  out,  that  tetanus  antitoxin 
has  been  disappointing  as  a  curative  agent.  Used  prophylactically 
it  is  very  much  more  satisfactory.  Flooding  the  nerves  near  the  site 
of  inoculation  with  antitoxin,  or  the  intracerebral  injection  of  anti- 
toxin in  desperate  cases  is  sometimes  successful.2  Tetanus  antitoxin 
has  also  been  administered  intraneurally  and  subdurally  in  desperate 
cases.  Subcutaneous  injection  is  comparatively  inefficient.  The  sub- 
cutaneous injection  of  two  hundred  or  more  units  at  the  site  of  infec- 
tion, or,  better,  after  exposure  of  the  regional  nerves,  is  said  to  be 
very  efficient  in  preventing  the  development  of  tetanus.  Calmette 
has  used  dried  tetanus  antitoxin  to  dust  the  navel  of  the  newborn  in 
the  tropics  and  the  deaths  from  tetanus  neonatorum  have  been  very 
greatly  reduced  by  this  procedure.3  Bockenheimer  has  made  a  dressing 
composed  of  an  ointment  mixed  with  tetanus  antitoxin,  which  is  also 
said  to  be  very  efficient  not  only  for  the  treatment  of  the  umbilicus 
of  the  newborn,  but  for  other  wounds  as  well. 

Tetanus  antitoxin  is  less  efficient  than  the  diphtheria  antitoxin  for 
several  reasons.  First,  the  diphtheria  antitoxin  has  a  greater  affinity 
for  its  toxin  in  vitro  than  the  tetanus  antitoxin  has  for  tetanus  toxin. 
Second,  diphtheria  toxin  appears  to  infect  principally  the  parenchy- 
matous  and  lymphatic  organs.  The  cells  comprising  these  organs  are 
less  susceptible  to  toxin  than  are  nerve  cells,  which  are  energetically 
attacked  by  tetanus  toxin.  The  diphtheria  toxin  has  less  affinity  for 
parenchymatous  cells  than  it  has  for  its  antitoxin,  and  the  diphtheria 
toxin,  furthermore,  circulates  in  the  blood  stream  where  the  antitoxin 
also  circulates  when  it  is  injected.  Treatment,  therefore,  with  diph- 
theria toxin  is  successful  even  after  symptoms  develop.  Fourth, 
tetanus  toxin  has  a  considerably  greater  affinity  for  nerve  cells  than 
it  has  for  its  own  antitoxin.  The  tetanus  antitoxin  is  "picked  up" 
by  the  end-organs  of  the  nerves  and  reaches  the  central  nervous  system 
by  the  axis  cylinders,  while  the  antitoxin  circulates  in  the  blood  and 
is  not  carried  to  the  central  nervous  system  by  way  of  the  nerves. 
Treatment  with  tetanus  antitoxin,  consequently,  is  rarely  successful 
after  symptoms  appear  and  practically  never  successful  after  the 
symptoms  have  been  developed  for  twenty-four  hours. 

1  Bull.  Johns  Hopkins  Hosp.,  July,  1895. 

2  Roux  and  Borrel,  Ann.  Inst.  Past.,  1898,  No.  4.     Chauffard  and  Quenu,  La  Presse 
Med.,  1898,  No.  5. 

3  It  must  be  remembered  that  the  albuminous  substances  contained  in  the  antitoxin, 
mixed  with  serum  from  the  wound,  make  a  favorable  culture  medium  for  many  bacteria ; 
the  dressings  must  be  sterile  and  watched  carefully  to  safeguard  the  patient. 


BACILLUS  TETANI  481 

The  Tetanus  Antitoxin  Unit. — The  tetanus  antitoxin  unit  of  the 
United  States  may  be  defined  as  "ten  times  the  minimal  quantity  of 
tetanus  antitoxin  necessary  to  protect  a  350-gram  guinea-pig  against 
a  standard  dose  of  tetanus  toxin  obtained  from  the  United  States 
Public  Health  and  Marine  Hospital  Laboratory."  It  has  theoretically 
the  power  to  neutralize  one  thousand  minimal  lethal  doses  of  tetanus 
toxin,  and  it  has,  consequently,  ten  times  the  theoretical  strength  of 
the  diphtheria  antitoxin  unit. 

Distribution  of  Tetanus  Bacilli  in  Nature. — Under  ordinary  con- 
ditions the  tetanus  bacillus  appears  to  be  a  saprophyte,  and  man  is 
not  necessary  for  its  continued  existence.  The  organisms  are  found 
very  commonly  in  the  excrement  of  the  herbivora,  notably  horses  and 
cattle.1  Sormani2  has  even  claimed  that  the  virulence  of  the  tetanus 
bacillus  is  maintained  by  frequent  passages  of  the  organism  through 
the  intestines  of  the  herbivora.  Pizzini3  has  found  tetanus  bacilli 
in  the  feces  of  peasants  who  tended  horses.  Not  all  observers,  however, 
subscribe  to  the  intestinal  theory.  Hoffmann,4  for  example,  found 
the  organism  only  once  out  of  twenty-two  samples  of  feces  from  twenty- 
two  different  horses. 

Tetanus  spores  are  found  widely  distributed  in  nature,  particularly 
in  the  upper  layers  of  the  soil;  in  temperate  climates  their  distribution 
is  somewhat  irregular,  but  in  the  tropics  they  appear  to  be  very  widely 
disseminated.  Tetanus  spores  also  occur  in  gelatin  occasionally,  and 
they  have  even  been  detected  in  cat  gut.  Levy  and  Bruns,5  and 
Anderson6  have  all  found  tetanus  spores  in  commercial  gelatin.  The 
potential  dangers  attending  the  use  of  gelatin  as  a  hemostatic  are 
apparent.7  Tetanus  spores  have  also  been  found  in  vaccine  virus  in 
the  past,8  and  Carini9  has  found  spores  in  vaccine  virus;  and  at  least 
two  outbreaks  of  tetanus,  one  in  this  country  and  one  in  Europe,  have 
resulted  from  the  infection  of  diphtheria  antitoxin  with  tetanus  spores. 
Rabinovitch10  has  also  found  tetanus  spores  in  washings  from  straw- 
berries sold  in  Berlin. 


1  Sanchez,  Toledo,  and  Baillon,  La  Semaine  Med.,  1,890,  No.  45;    Centralbl.  f.  Bakt. 
1890,  ix,  18. 

2  Behand.  der  10th  Intern,  med.  Kong.,  Berlin,  1890,  v,  152. 

3  Riv.  d'igiene  e  san.  publ.,  1898,  x,  170. 

4  Hyg.  Rund.,  1905,  xv,  1233. 

5  Grenzgeb.  der  Med.  u.  Chir.,  1902,  x,  235;   Deutsch.  med.  Wchnschr.,  1902,  130. 

6  Mar.  Hosp.  Lab.  Bull.,  1902,  ix. 

7  Zibell,  Munchen.  med.  Wchnschr.,  1901,  1643,  for  literature. 

8  McFarland,  Lancet,  September,  1902. 

9  Centralbl.  f.  Bakt.,  Orig.,  1904,  xxxvii,  48. 

10  Arch.  f.  Hyg.,  1907,  Ixi,  103. 

31 


482  ANAEROBIC  BACTERIA 

Pathogenesis. — Tetanus  occurs  spontaneously  in  man,  horses,  cattle 
and  sheep,  rarely  in  dogs  and  goats.  Birds  and  reptilia  are  highly 
refractory  to  experimental  inoculation.  The  disease  tetanus  both  in 
man  and  animals  is  purely  toxic  in  character;  notwithstanding 
the  wide  distribution  of  tetanus  spores,  it  is  relatively  uncommon. 
It  may  follow  traumatism,  particularly  deep,  narrow  wounds  and  con- 
tused wounds  to  which  tetanus  spores,  together  with  other  organisms 
gain  entrance.  In  the  tropics  an  infection  of  the  umbilicus  of  the 
newborn  (tetanus  neonatorum)  is  very  common.1  Postpartum  infec- 
tions, particularly  of  the  uterus  (tetanus  puerperalis),  were  also  at 
one  time  very  common.2 

The  lesions  observed  in  tetanus  are  very  slight  and  postmortem 
there  may  be  no  marked  changes  other  than  a  slight  congestion  of 
the  internal  organs.  Bacilli  may  occasionally  be  found  at  the  site  of 
inoculation,  but  they  do  not  as  a  rule  penetrate  deeply  into  the  body, 
although  Hochsinger3  and  Creite4  have  found  the  organisms  at  autopsy 
in  a  very  few  instances  in  the  spleen  and  heart  blood. 

Tarozzi5  and  Canfora6  have  studied  the  fate  of  tetanus  spores  after 
subcutaneous  inoculation  into  guinea-pigs  and  rabbits  very  carefully. 
They  find  the  spores  may  be  transmitted  rather  rapidly  to  the  paren- 
chymatous  organs,  liver,  spleen,  and  kidneys  principally,  where  they 
may  remain  alive  but  latent  for  seven  to  eight  weeks.  If  trauma 
or  injury  resulting  in  inflammation  occurs  during  this  time,  acute 
or  chronic  tetanus  may  result.  These  observations  suggest  a  possible 
explanation  for  the  so-called  cryptogenetic,  ideopathic,  or  rheumatic 
tetanus;  the  intestinal  tract  is  supposed  to  be  an  occasional  portal 
of  entry,  thus  explaining  another  source  of  cryptogenetic  tetanus. 

Experimental  Pathogenesis  in  Animals. — The  disease  tetanus  may 
be  produced  in  susceptible  animals  by  injecting  soil  or  active  cultures 
of  tetanus  bacilli,  spores  mixed  with  tetanus  toxin,  or  tetanus  toxin 
alone.  If,  however,  tetanus  spores  carefully  freed  from  toxin  are 
injected  alone,  tetanus  frequently  fails  to  develop.  Vaillard  and 
Vincent7  and  Vaillard  and  Rouget8  have  furnished  an  interesting 


1  Anders  and  Morgan,  Jour.  Am.  Med.  Assn.,  1906,  xlvii,  2083. 

2  Stern,  Deutsch.  med.  Wchnschr.,  1892,  No.  12.     Heyse,  Berl.  klin.  Wchnschr.,  1893, 
No.  24. 

3  Centralbl.  f.  Bakt.,  1887,  ii,  145.     Hohlbeck,  Deutsch.  med.  Wchnschr.,  1903,  172. 

4  Centralbl.  f.  Bakt.,  Orig.,  1904,  xxxvii,  312. 
6  Ibid.,  1905,  xxxviii,  619. 

6  Ibid.,  1908,  xlv,  495. 

7  Ann.  Inst.  Past.,  1891,  24. 

8  Ann.  Inst.  Past.,  1892,  428;    Centralbl.  f.  Bakt.,  xvi,  208. 


BACILLUS   TETANI  483 

explanation  for  this  possibility.  They  find  that  phagocytosis  plays 
an  important  part  in  the  removal  of  tetanus  spores  which  are  injected 
without  tetanus  toxin  or  other  irritating  substances.  Polymorpho- 
nuclear  leukocytes  engulf  free  tetanus  spores.  If,  however,  the  spores 
are  introduced  into  the  body  in  collodion  capsules,  thus  protecting 
the  organisms  from  the  leukocytes,  the  tetanus  spores  develop  into 
bacilli  there,  form  toxin,  and.  produce  tetanus.  If  tetanus  spores  are 
mixed  with  lactic  acid,  with  tetanus  toxin,  or  with  other  irritants,  or 
even  injected  with  saprophytic  bacteria,  the  spores  develop  into  tetanus 
bacilli,  produce  toxin  and  kill  the  animal. 

Bacteriological  Diagnosis. — 1.  Microscopical. — Smears  made  from 
the  pus  of  wounds  in  suspected  cases  of  tetanus  may  show  the  charac- 
teristic spores  of  the  tetanus  bacilli.  The  organisms,  however,  are 
usually  present  in  very  small  numbers  and  several  smears  should  be 
made.  Negative  results  do  not  prove  the  absence  of  the  tetanus 
bacillus. 

2.  Cultural. — Pus  from  wounds  scraped  out  with  sterile  curettes, 
or  suspected  material  is  placed  in  fermentation  tubes  containing  bits 
of  sterile  tissue,  according  to  Theobald  Smith's  method  mentioned 
above,  incubated  for  forty-eight  hours  and  examined  -microscopically 
for  typical  spores.     If  these  are  found  the  material  is  heated  to  80° 
C.  for  thirty  minutes  to  kill  vegetative  forms  and  then  reinoculated 
to  obtain  growths  of  the  organism. 

3.  Toxin. — Inoculation  of  material  containing  tetanus  bacilli  and 
other  organisms  into  slightly  alkaline  broth  (sugar-free)  grown  anae- 
robically  for  six  or  eight  days  will  lead  to  toxin  formation  even  if 
other  bacteria  are  present.    Inoculation  of  this  toxic  broth  into  mice 
will    frequently    give    positive    results.      Broth   obtained    according 
to  the  Theobald  Smith  method  in  Step  2  also  should  be  inoculated 
into  mice  if  the  preliminary  microscopic  examination  shows  tetanus 
spores. 

4.  At  times  tetanus  toxin  occurs  even  in  the  blood  of  the  patient, 
provided  no  antitoxin  has  been  administered;    1  c.c.  of  this  blood 
inoculated  into  a  mouse  may  occasionally  produce  characteristic  tetanic 
phenomena. 

Prophylaxis. — Any  wound  likely  to  be  a  suitable  portal  of  entry  for 
the  tetanus  bacillus  should  be  regarded  as  potentially  dangerous  and 
tetanus  antitoxin  should  be  administered  promptly  as  a  prophylactic 
measure.  Fifteen  hundred  units  of  tetanus  antitoxin  is  the  ordinary 
prophylactic  dose  in  such  cases,  For  curative  doses  3000  to  20,000 


484  ANAEROBIC  BACTERIA 

units  have  been  injected  locally,  intraneurally  or  subdurally,  depend- 
ing upon  the  condition  of  the  patient  and  the  time  which  has  elapsed 
since  infection  took  place.  The  results  are  usually  unsatisfactory  if 
symptoms  of  tetanus  have  developed,  but  the  treatment  should  be 
carried  out  energetically. 

BOTULISM   OR   ALLANTIASIS. 

A  rather  definite  train  of  symptoms  consisting  of  gastro-intestinal 
irritation,  nervous  disturbances,  bulbar  paralysis,  dysplagia  and 
protrusion  of  the  eyeballs  with,  however,  no  fever,  has  occasionally 
followed  the  consumption  of  uncooked  or  imperfectly  cooked  meats 
or  fish.  Uncooked  products,  particularly  ham  and  sausages,  are 
more  commonly  the  source  of  these  intoxications.  The  mortality  is 
fairly  high  in  such  cases,  amounting  to  as  much  as  25  per  cent,  in 
various  epidemics.  Patients  retain  consciousness  to  the  end  as  a 
rule. 

The  best-studied  epidemic  of  this  type  was  one  which  occurred  in 
Ellezelles,  Belgium.  Von  Ermengem1  investigated  this  epidemic  very 
thoroughly  and  found  that  all  the  cases  had  partaken  of  an  imper- 
fectly cured  ham,  from  which  he  isolated  an  organism  which  he  called 
B.  botulinus.  He  established  the  relationship  of  the  organism  to  the 
disease  which  resulted  from  the  ingestion  of  the  toxins  of  this  bacillus 
by  animal  experimentation. 

Morphology. — Bacillus  botulinus  is  a  rather  large  bacillus,  measuring 
from  0.9  to  1.2  microns  in  diameter  by  4  to  6  microns  in  length,  with 
rounded  ends;  it  occurs  singly  or  in  pairs,  less  commonly  in  short 
chains  of  three  to  six  elements.  Old  cultures  of  this  organism  and  those 
incubated  above  36°  C.  show  involution  forms  which  are  usually  long, 
intertwined  filaments.  The  organism  is  sluggishly  motile  and  has 
from  4  to  8  peritrichic  flagella.  It  forms  oval  spores,  slightly  greater 
in  diameter  than  the  rod  and  situated  near  one  end  of  it.  The  organism 
stains  readily  with  anilin  dyes  and  is  Gram-positive. 

Isolation  and  Culture. — Bacillus  botulinus  grows  most  characteris- 
tically in  slightly  alkaline  dextrose  gelatin  incubated  at  25°  C.  under 
strictly  anaerobic  conditions.  The  colonies,  which  grow  with  moderate 
rapidity,  are  light  yellow  in  color,  nearly  transparent,  and  are  com- 
posed of  coarse  granules.  These  granules  after  a  few  hours'  growth 
exhibit  a  slow  but  constant  motion  in  a  zone  of  liquefied  gelatin.  As 

1  Centralbl,  f.  Bakt.,  1896,  xix,  442;   Ztschr.  f.  Hyg.,  1897,  xxvi,  1. 


BOTULISM  OR  ALLANTIASIS  485 

they  reach  their  maximum  development  the  colonies  become  brown 
and  opaque,  and  only  those  granules  at  the  periphery  of  the  colony 
remain  motile. 

Growth  in  Artificial  Media — The  organism  grows  well  in  the  ordinary 
nutrient  media,  better  when  dextrose  is  added,  but  only  under  anae- 
robic conditions.  A  strong  odor  of  butyric  acid  is  characteristic  of 
growths  of  the  organism  in  artificial  media.  It  is  essential  to  transfer 
large  amounts  of  material  to  insure  growth  of  the  organism.  Gelatin 
is  liquefied.  The  growth  on  agar  is  very  similar  to  that  in  gelatin, 
except  that  no  liquefaction  takes  place  and  no  motile  granules  appear 
in  the  colonies.  A  slight  turbidity  is  developed  in  plain  broth  after 
twenty-four' hours'  incubation,  a  heavy  turbidity  in  dextrose  broth. 
The  organism  grows  well  in  milk,  producing  a  slightly  acid  reaction 
but  neither  coagulation  nor  peptonization. 

The  organism  is  an  obligate  anaerobe,  whose  optimum  temperature 
of  growth  is  22°  to  25°  C.  It  grows  but  slowly  at  25°  C.  Incubation 
at  the  latter  temperature  leads  to  the  development  of  involution  forms 
and  an  inhibition  of  spore  formation  and  toxin  production.  The 
spores  are  not  particularly  resistant  to  heat  or  disinfectants  and 
cultures  die  out  in  three  to  four  weeks  unless  transferred  to  fresh 
media  within  that  time.  The  spores  are  killed  by  an  exposure  at  80°  C. 
for  sixty  minutes.  Five  per  cent,  carbolic  acid  kills  them  in  twenty- 
four  hours  and  pickling  in  10  per  cent,  salt  solution  kills  them  within 
a  week.  If  the  spores  are  protected  from  oxygen  and  sunlight  they 
retain  their  vitality  for  several  months,  either  in  a  moist  condition  or 
dried. 

Products  of  Growth — The  organism  produces  an  active  soluble 
gelatinase  in  plain  broth  cultures  and  in  gelatin,  particularly  the 
latter.  It  forms  acid  and  gas  in  dextrose  broth;  bioses  and  polysac- 
charides  are  not  fermented.  The  acid  formed  is  partly  butyric,  and  the 
gas  consists  principally  of  carbon  dioxide  and  hydrogen. 

The  most  important  product  of  B.  botulinus,  however,  is  a  potent 
extracellular  toxin  which  is  readily  prepared  by  growing  the  organisms 
anaerobically  in  sugar-free  broth  at  25°  C.  for  two  weeks.  The 
broth  is  filtered  through  sterile  porcelain  filters,  preferably  in  an  atmos- 
phere of  hydrogen,  and  the  toxin  is  found  in  the  filtrate,  from  which 
it  can  be  precipitated  by  the  addition  of  a  3  per  cent,  aqueous  solution 
of  zinc  chloride  in  the  proportions  of  two  parts  of  zinc  chloride  to 
one  of  broth.1  The  toxin  deteriorates  rather  rapidly  if  it  is  exposed 

1  Brieger  and  Kempner,  Deutsch.  med.  Wchnschr.,  1897,  xxxiii,  521. 


486  ANAEROBIC  BACTERIA 

to  sunlight  or  oxygen.  If  it  is  kept  in  the  dark  in  sealed,  full  bottles 
and  kept  cool  it  retains  its  potency  for  some  months.  It  keeps  still 
better  dried  in  the  absence  of  light  and  moisture.  Heat  promptly 
inactivates  it.  An  exposure  at  58°  C.  for  three  hours,  or  at  80°  C.  for 
thirty  minutes  utterly  destroys  its  potency.  It  is  not,  however, 
destroyed  by  putrefaction  or  by  gastric  digestion,  a  point  of  great 
importance  clinically,  for  poisoning  with  the  toxin  of  B.  .botulinus 
almost  always  results  from  its  absorption  from  the  intestinal  tract. 
The  toxin  has  also  been  isolated  from  hams  in  which  the  organisms 
have  grown.  The  hams  are  macerated  with  water  in  a  cool,  dark 
place,  filtered  through  porcelain,  and  the  filtrate  is  found  to  contain 
the  toxin.  The  toxin  also  is  produced  when  the  organisms  grow  under 
proper  conditions  in  vegetables.1  The  toxin  causes  death  when 
injected  subcutaneously  or  fed  to  experimental  animals.  There  is  a 
latent  period  which  elapses  between  the  time  of  administration  of  the 
toxin  and  the  appearance  of  symptoms.  This  latent  period  when  large 
doses  are  administered  is  from  twelve  to  twenty  hours;  with  moderate 
doses  it  is  about  thirty-six  hours.  One-thousandth  c.c.  of  broth  con- 
taining toxin  injected  subcutaneously  into  guinea-pigs  usually  kills  them 
in  three  to  four  days;  0.1  to  0.5  c.c.  of  the  same  toxin  absorbed  in 
bread  and  fed  to  rabbits  results  fatally  in  from  four  to  six  days.  It 
is  toxic  for  man,  white  rats,  mice,  kittens,  guinea-pigs,  rats,  and  even 
monkeys  in  relatively  small  doses.  In  larger  doses  it  is  also  patho- 
logical for  cats  and  doves.  The  toxin  is  bound  by  the  gray  matter 
of  the  central  nervous  system.  Cholesterin,  lecithin,  and  fats  such 
as  butter  and  oils  are  believed  to  bind  the  toxin  as  well. 

Antitoxin. — Kempner2  has  succeeded  in  immunizing  goats  to  the 
toxin  of  B.  botulinus,  and  has  identified  in  their  serum  a  specific  anti- 
toxin which  has  considerable  potency  both  curatively  and  prophylac- 
tically.  Wassermann  has  been  able  to  immunize  horses  with  the  same 
results.  The  antitoxin  neutralizes  the  toxin  both  in  vivo  and  in  vitro.3 
Leuchs  has  shown  that  dilute  acids  will  split  up  the  toxin-antitoxin 
combination  into  the  two  components,  both  of  which  may  be  recovered. 

Pathogenesis — The  lesions  produced  by  the  toxin  both  in  man  and 
in  animals  are  very  similar,  and  the  symptoms  produced  are  referable 
to  the  action  of  the  toxin  on  the  medulla  and  cord.4  There  is  bulbar 
paralysis,  paralysis  of  the  eye  muscles,  great  muscular  weakness, 

1  Landmann,  Hyg.  Rundschau,  1894,  449. 

2  Ztschr.  f.  Hyg.,  1897,  xxvi,  482. 

3  Forssman  and  Lundstrom,  Ann.  Inst.  Past.,  1902,  294. 

4  Kempner  and  Scheplewsky,  Ztschr.  f.  Hyg.,  1898,  xxvii,  214. 


BOTULISM  OR  ALLANTIASIS  487 

profuse  nasal  and  oral  discharge,  aphagia,  aphonia,  and  interference 
with  the  workings  of  the  cardiac  and  respiratory  centres.  Micro- 
scopically there  are  degenerative  changes  limited  chiefly  to  the  cells 
of  the  gray  matter  of  the  medulla,  cord  and  salivary  glands.1 

The  disease  produced  by  ingestion  or  injection  of  toxins  of  B.  botu- 
linus  in  experimental  animals  reproduces  faithfully  the  symptom-com- 
plex seen  in  the  naturally  acquired  disease  in  man.  The  organism 
itself  does  hot  appear  to  grow  in  the  tissues  of  warm-blooded  animals 
except  just  before  and  after  death,  hence  it  is  logical  to  conclude  that 
the  ingestion  of  food  containing  the  toxins  of  this  organism  rather 
than  the  generation  of  the  toxin  in  the  tissues  of  the  host  is  the  source 
of  intoxication. 

Bacteriological  Diagnosis. — The  bacteriological  diagnosis  can  not 
be  made  ordinarily  in  man.  It  is  necessary  in  the  vast  majority  of 
instances  to  obtain  the  meat  in  which  the  organisms  have  grown. 

(a)  Microscopic. — This  is  usually  not  feasible. 

(b)  Cultural. — Make   anaerobic   dextrose   gelatin   plates   from   the 
suspected  meat,  selecting  portions  which  are  removed  from  contami- 
nated surfaces,  as  follows:     (1)  Rapidly  make  a  maceration  of  some 
of  the  meat  in  sterile  salt  solution.     (2)  Heat  some  of  the  opalescent 
fluid  to  60°  C.  for  thirty  minutes,  and  make  plates.    (3)  Add  some  of 
the  opalescent  fluid  to  fermentation  tubes  according  to  Theobald 
Smith's  method   (see  page  473)  with  bits  of  sterile  animal  tissue. 
(4)  Plate  some  of  the  opalescent  fluid  directly  without  heating  into 
dextrose  gelatin  plates.     (5)  Examine  the  media  for  characteristic 
colonies. 

(c)  Identification  of  Toxin. — 1.  Filter  some  of  the  macerated  meat 
rapidly  through  sterile  filter  paper  and  inject  0.5  to  1  c.c.  subcu- 
taneously  into  a  rabbit  or  guinea-pig.    The  protruding  eyeballs  and 
respiratory  failure  usually  suffice  to  establish  the  diagnosis,  which 
may  be  confirmed  by  staining  sections  of  the  central  nervous  system 
and  identifying  the  lesions.    (2)  Add  2  to  5  c.c.  of  the  filtrate  to  some 
bread  and  feed  a  rabbit  with  it.     Note  the  symptoms.     (3)  Filter 
some  of  the  broth  from  the  fermentation  tube  in  Step  3  of  the  cultural 
identification  and  inject  subcutaneously  or  feed  to  a  rabbit  and  observe 
symptoms. 

(d)  Inspection  of  Suspected  Meat. — It  is  difficult  usually  to  detect 
anything  abnormal  in  meat  in  which  B.  botulinus  has  grown.     Occa- 

1  Marineseo,  Compt.  rend.  soc.  de  biol.,  1896.  Kempner  and  Pollak,  Deutsch.  med. 
Wchnschr.,  1897,  xxxiii,  521. 


488  ANAEROBIC  BACTERIA 

sionally  a  slight  odor  of  butyric  acid  is  noticed;  usually  there  is  no 
sign  recognizable  either  by  smell  or  taste  which  will  furnish  a  clue  to 
the  unfitness  of  the  meat  for  food. 

Prophylaxis. — The  disease  is  not  contagious  and  patients  are  not  a 
source  of  danger  to  others.  The  organisms  are  not  as  a  rule  found  in 
man.  The  toxin  is  thermolabile;  consequently  thorough  cooking  of 
foods  will  eliminate  all  danger.  Hams,  similar  meats  and  meat  pro- 
ducts alone  cause  the  disease.  If  such  meats  are  cured  by  pickling 
they  should  be  immersed  in  the  pickle  not  less  than  a  week  and  the 
pickle  should  contain  the  equivalent  of  10  per  cent,  salt  solution. 

BACILLUS   AEROGENES   CAPSULATUS. 

Historical.1 — This  organism  was  first  described  by  Welch  in  1891, 
and  later  in  detail  by  Welch  and  Nuttall.2  It  appears  to  be  identical 
with  Bacillus  phlegmonis  emphysematosaB,3  B.  perfringens,4  B.  emphy- 
sematis  vaginae,5  and  possibly  B.  enteritidis  sporogenes6  and  Granulo- 
bacillus  saccharo  butyricus  immobilis  liquefaciens.7  It  is  commonly 
referred  to  as  the  "gas  bacillus."  The  organism  has  been  described 
most  commonly  in  the  past  as  the  causative  agent  of  the  so-called 
"foamy  organs."  It  was  isolated  by  Welch  from  such  a  case  in  1891, 
and  it  has  been  isolated  many  times  since  from  similar  lesions. 

Morphology. — B.  aerogenes  capsulatus  is  a  rather  large  bacillus, 
measuring  from  1  to  1.2  microns  in  diameter  and  from  2  to  5  microns 
in  length,  with  somewhat  square-cut  ends,  occurring  usually  singly 
or  in  pairs;  in  artificial  culture  media  rarely  in  short  chains.  Accord- 
ing to  Welch,  the  organism  tends  to  form  chains  in  bloodvessels.  The 
organisms  under  these  conditions  may  be  somewhat  shorter  than 
those  typically  found  in  artificial  media,  frequently  being  but  1.5 
to  2  microns  in  length. 

The  organism  is  non-motile  and  possesses  no  flagella.  It  forms 
capsules  in  the  animal  body  and  occasionally  in  albuminous  media. 
It  also  forms  spores,  first  observed  by  Dunham.8  The  spores  are 

1  For  an  excellent  study  and  critical  summary  see  Simonds,  Monograph  V,  Rockefeller 
Institute  for  Medical  Research,  September  27,  1915. 

2  Johns  Hopkins  Bull.,  1892,  iii,  81. 

3  Frankel,  Centralbl.  f.  Bakt.,  1893,  xiii,  13. 

4  Veillon  and  Zuber,  Arch,  de  med.  exper.  et  d'anat.  path.,  1898,  x,  517. 

5  Lindenthal,  Wien.  klin.  Wchnschr.,  1897,  x,  3. 

6  Klein,  Centralbl.  f.  Bakt.,  1895,  xviii,  737. 

7  Schattenfroh  and  Grassberger,  Centralbl.  f.  Bakt.,  ii  abt.,  1899,  v,  209;     Miinchen. 
med.  Wchnschr.,  1900,  Nos.  30-31;    Wien.  klin.  Wchnschr.,  1900,  No.  48. 

8  Johns  Hopkins  Bull.,  1897,  viii,  68. 


BACILLUS  AEROGENES  CAPSULATUS  489 

oval,  somewhat  less  in  diameter  than  the  vegetative  form  of  the 
organism,  and  are  usually  situated  near  one  end  of  the  rod.  But  one 
spore  is  found  in  a  single  organism.  Spores  are  apparently  not  formed 
in  the  tissues  of  the  body.  The  organism  stains  readily  with  ordinary 
anilin  dyes.  It  is  Gram-positive,_although  old  cultures  on  artificial 
media  exhibit  irregularities  in  staining,  probably  due  to  beginning 
degeneration. 

Isolation  and  Culture. — The  organism  is  an  obligate  anaerobe.  It 
grows  well  in  all  ordinary  media  containing  dextrose  or  lactose.  From 
tissues  it  is  best  obtained  on  anaerobic  agar  plates,  where  the  colonies 
are  round,  semi-translucent  and  colorless,  and  not  characteristic. 
Many  strains  hemolyze  blood  and  on  blood  agar  the  colonies  are  sur- 
rounded by  a  rather  narrow  zone  of  hemolysis.  From  the  intestinal 


; 

FIG.  66. — Bacillus  aerogenes  capsulatus  from  pure  milk  culture.      X  1000. 


contents  the  organism  is  best  isolated  in  milk.  A  thin  suspension  of 
feces  is  emulsified  in  milk  (whole  milk)  after  the  milk  has  been  boiled 
and  rapidly  cooled  to  remove  all  oxygen.  The  milk  is  heated  to  80° 
C.  for  twenty  minutes  to  kill  vegetative  organisms,  and  then  it  is 
incubated  at  body  temperature  for  eighteen  to  twenty-four  hours. 
At  the  end  of  that  time  the  milk  exhibits  a  characteristic  stormy  fer- 
mentation. The  casein  is  reduced  in  amount  and  the  residual  casein 
is  full  of  holes  and  is  usually  slightly  pink  in  color.  The  whey  is  usually 
colorless,  gas  bubbles  are  seen  at  the  top  of  it,  and  there  is  character- 
istically an  odor  of  rancid  butter — butyric  acid.  The  organism  may  be 
obtained  from  the  milk  culture  directly  by  plating  anaerobically  on 
agar,  or  it  may  be  obtained  by  injecting  some  of  the  whey  into  the 
ear  vein  of  a  rabbit,  killing  the  animal  after  five  minutes  and  incubating 


490  ANAEROBIC  BACTERIA 

it  for  twelve  to  eighteen  hours.1  The  rabbit  will  be  found  to  be  enor- 
mously distended  with  gas.  The  tissues,  particularly  the  muscles, 
will  be  found  to  be  soft,  partly  liquefied,  and  the  course  of  the  blood- 
vessels will  be  marked  out  by  rows  of  gas  bubbles.  The  organisms  are 
found  in  greatest  abundance  in  the  liver,  which  is  light  colored2  and 
in  typical  cases  so  thoroughly  fermented  that  it  appears  to  be  a  collec- 
tion of  gas  bubbles.  The  gas  bubbles  found  in  the  blood  stream  and 
in  the  muscles  and  particularly  in  the  liver  are  the  result  of  the 
decomposition  of  the  muscle  sugar  and  glycogen  by  this  organism. 


FIG.  67. — Bacillus  aerogenes  capsulatus,  capsule  stain.      X  1000. 

Growth  on  Artificial  Media. — Anaerobic  growth  on  gelatin  is  variable ; 
some  strains  do  not  grow  in  this  medium,  others  produce  a  slight 
liquefaction.  In  plain  broth  there  is  a  slight  turbidity;  in  broth  con- 
taining dextrose  or  lactose  the  turbidity  is  marked.  The  reaction  in 
milk  has  been  described  previously,  the  characteristic  features  being 
a  stormy  fermentation,  a  slight  pink  color  to  the  undissolved  casein, 
and  gas  bubbles  together  with  a  slight  odor  of  butyric  acid.  If  the 
milk  has  not  been  heated  sufficiently  to  remove  all  oxygen  the  organism 
frequently  produces  coagulation,  but  no  stormy  fermentation  and  no 
gas. 

Conditions  of  Growth. — The  organism  is  an  obligate  anaerobe  which 
does  not  grow  below  20°  C.,  or  above  45°  C.  The  optimum  tempera- 
ture of  growth  is  37.5°  C.  The  spores  are  quite  resistant;  five  minutes' 
boiling  usually  fails  to  kill  them.  They  are  extremely  resistant  to 

1  This  procedure  is  frequently  known  as  the  "Welch  Nuttall  Test." 

2  The  absence  of  darkening  of  the  liver  tissue  indicates  that  little  or  no  proteolysis 
is  taking  place;  otherwise  the  liver  would  be  discolored,  due  to  the  production  of  sul- 
phide of  iron  from  the  liberation  of  H2&  of  protein  and  its  reaction  upon  the  blood. 


BACILLUS  AEROGENES  CAPSULATUS  491 

drying,  particularly  in  the  absence  of  sunlight.  Viable  spores  have 
been  obtained  from  dust  in  a  vault  which  had  not  been  opened  for 
fifteen  years.  Sporulation  does  not  take  place,  as  a  rule,  in  the  tissues; 
spores  are  frequently  found  in  the  intestinal  tract.  They  do  not  form 
readily  in  media  containing  utilizable  carbohydrates,  but  are  found 
on  the  surface  of  slanted  blood  serum1  and  in  protein  media.  Simonds2 
finds  that  an  acidity  greater  than  1  per  cent,  to  phenolphthalein 
inhibits  spore  formation. 

Products  of  Growth. — B.  aerogenes  capsulatus  forms  a  gelatinase 
in  the  absence  of  utilizable  sugars.  In  dextrose,  lactose  and  saccharose 
media  it  produces  an  energetic  fermentation,  the  products  being 


FIG.  68. — Bacillus  aerogenes  capsulatus,  smear  from  liver  of  rabbit.     X  1000. 

butyric  and  lactic  acids,  carbon  dioxide  and  hydrogen  in  the  pro- 
portions H:CO2  =  i  approximately;3  comparatively  little  acid  is 
formed.  Welch  and  Nuttall  state  that  the  organism  decomposes 
protein  with  the  formation  of  carbon  dioxide  and  hydrogen  and  nitro- 
gen gas;  but  it  is  probable  that  little  or  no  gas  is  formed  from  protein. 
According  to  Brown,4  the  organism  forms  a  toxin  in  sugar-free  broth, 
which  is  pathogenic  for  guinea-pigs.  The  toxin  is  not  formed  in  broth 
containing  utilizable  carbohydrates. 

Simonds5  has  distinguished  four  distinct  types  or  subgroups  of 
Bacillus  aerogenes  capsulatus,  which  differ  essentially  in  their  fer- 
mentation of  certain  sugars  and  in  their  sporulation  as  follows : 

1  Dunham,  loc.  cit. 

2  Loc.  cit.,  p.  31. 

3  Smith,  Brown,  and  Walker,  Jour.  Med.  Research,  1905-1906,  xiv,  193. 

4  Annual  Report,  Massachusetts  State  Board  of  Health,  1909. 

5  Loc.  cit.,  p.  13. 


492 


ANAEROBIC  BACTERIA 


Fermentation.1 

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Pathogenesis. — The  pathogenesis  of  B.  aerogenes  capsulatus  is  very 
variable.  The  production  of  emphysematous  gangrene  in  contused 
wounds  and  compound  fractures  is  the  best  known  of  its  pathogenic 
properties.  According  to  Achalme,3  the  organism  has  been  isolated 
from  the  blood  stream  in  cases  of  acute  articular  rheumatism.  This 
observation  has  been  made  by  others  also.  It  has  not  been  proven, 
however,  that  the  organism  causes  acute  articular  rheumatism.  In 
the  intestinal  tract4  the  organism  occasionally  produces  disease  which 
varies  in  severity  from  a  mild  diarrhea  to  an  extremely  acute  dysenteric 
diarrhea.  Epidemics  of  such  diarrhea  appear  to  have  been  traced  in 
a  few  instances  to  milk.5  The  organism  appears  to  cause  an  intense 
irritation  in  the  intestinal  tract,  probably  due  to  the  production  of 
butyric  acid,  but  there  is  no  evidence  that  the  intestinal  infection  is 
a  true  toxemia.6  Usually  the  organism  is  not  invasive,  but  in  a  few 
cases  the  mucosa  of  the  large  intestine  has  been  distinctly  involved. 
The  mucosa  was  enormously  swollen  and  edematous  and  the 
organisms  were  found  deep  in  the  submucosa.  In  one  instance  at 
least  the  organism  has  been  isolated  from  tonsils  in  a  case  of  chronic 

1  Gas  and  acid. 

2  This  table  is  in  harmony  with  the  view  that  the  organism  does  not,  as  a  rule,  sporu- 
late  in  media  containing  utilizable  sugar.     It  is  probable  that  the  acid  products  of 
fermentation  inhibit  sporulation,  as  Simonds  has  shown. 

3Compt.  rend.,  Soc.  de  biol.,  1891,  xliii,  651;  1897,  xlix,  276;  Ann.  Inst.  Past., 
November  25,  1897. 

4  Howard  (Johns  Hopkins  Hosp.  Rep.,  1900,  ix,  461)  states  that  the  organism  may 
develop  in  the  gastric  or  intestinal  mucosa,  especially  under  the  folds  of  the  valvulse 
conniventes,  and  cause  disintegration  of  the  tissue. 

6  Klein,  Annual  Report  of  the  Medical  Officer  of  the  Local  Government  Board,  London, 
1897-1898,  No.  27,  p.  210. 

6  Kendall  and  Smith,  Boston  Med.  and  Surg.  Jour.,  1911,  clxiv,  306.  Kendall,  Day 
and  Bagg,  ibid.,  1913,  clxix,  741.  Kendall  and  Day,  ibid.,  753.  Kendall,  ibid.,  May 
20,  1915. 


BACILLUS  CEDEMATIS  MALIGNI  493 

hypertrophic  tonsillitis;  the  organisms  were  deep  in  the  tissues, 
where  they  could  be  distinctly  seen  in  sections,  and  they  did  not  form 
spores;  at  least  none  could  be  demonstrated  by  the  ordinary  methods. 
When  the  organism  was  isolated  from  the  tonsillar  tissue  it  appeared 
to  have  lost  its  fermentative  powers  to  a  very  considerable  degree, 
but  rapidly  regained  them  with  repeated  transfer  in  artificial  media. 
Similarly,  the  organism  has  been  isolated  from  the  petrous  portion 
of  the  temporal  bone  bilaterally  in  an  infant  which  died  of  a  severe 
gas  bacillus  infection  of  the  intestinal  tract. 

Distributions. — The  organism  is  found  in  sewage,  in  impure  water, 
in  dust,  very  frequently  in  the  intestinal  tract  of  man,  and  probably 
of  animals.  In  the  past  B.  aerogenes  capsulatus  has  almost  undoubt- 
edly been  confused  with  the  bacillus  of  malignant  edema.  Thus, 
Grigorjeff  and  Ukke1  found  an  organism  complicating  typhoid  fever, 
which  produced  typical  foamy  organs;  this  they  identified  as  the 
bacillus  of  malignant  edema.  It  is  very  probable  that  this  organ- 
ism was  in  reality  the  gas  bacillus,  as  was  the  organism  described  by 
Brieger  and  Ehrlich2  in  a  somewhat  similar  case. 

BACILLUS   CEDEMATIS   MALIGNI. 

Historical. — The  bacillus  of  malignant  edema  is  the  oldest  known 
anaerobic  organism  of  which  there  is  an  authentic  description.  It  was 
described  by  Pasteur,  Joubert  and  Chamberland,3  later  by  Koch.4 
Pasteur  and  his  associates  obtained  the  bacillus  from  a  localized  epi- 
demic of  acute  septicemia  in  small  animals,  characterized  by  a  local 
edema  at  the  site  of  infection.  They  reproduced  the  disease  by  inocu- 
lating the  organism  into  other  animals,  or  by  the  injection  of  putres- 
cerit  animal  tissues.  The  bacillus  was  called  Vibrion  septique.  Koch 
studied  malignant  edema  in  larger  animals  and  called  attention  to 
the  localized  edema  and  the  absence  of  generalized  sepsis,  which  are 
the  characteristic  features  of  the  disease.  Koch  called  the  organism 
Bacillus  oedematis  maligni. 

Morphology. — B.  oedematis  maligni  is  a  slender  rod,  0.8  to  1  micron 
in  diameter  by  2  to  10  microns  in  length,  with  rounded  ends,  frequently 
occurring  in  long  chains,  particularly  in  the  animal  body.  It  is  motile 
under  anaerobic  conditions  and  possesses  numerous  peritrichic  flagella, 
usually  about  twenty.  No  capsule  has  been  observed.  Sporulation 

1  Centralbl.  f.  Bakt.,  1899,  xxv,  253.  2  Berl.  klin.  Wchnschr.,  1882,  No.  44. 

3  Bull,  de  1'acad.  de  Science,  1878,  Ixxxvi,  1038. 

4  Mitt.  a.  d.  kais.  Gesamte,  1881,  i,  52. 


494  ANAEROBIC  BACTERIA 

takes  place  readily,  and  the  spores  occur  typically  in  the  centre  of 
the  rod,  giving  it  a  slightly  swollen  appearance.  The  organism  stains 
readily  with  ordinary  anilin  dyes  and  is  usually  regarded  to  be  Gram- 
negative,  although  some  claim  it  is  Gram-positive.1 

Isolation  and  Culture. — B.  cedematis  maligni  is  a  strict  anaerobe, 
and  the  organisms  are  best  obtained  in  pure  culture  from  the  edema- 
tous  lesions  produced  in  rabbits  or  in  guinea-pigs  by  inoculation  of 
them  with  garden  soil.  The  organism  grows  readily  under  anaerobic 
conditions  on  dextrose  agar,  and  the  colonies  produced  are  very 
filamentous. 


FIG.  69. — Bacillus  cedematis  maligni,  spore  formation.      X  1000.     (Kolle  and  Hetsch.) 

The  organism  grown  anaerobically  on  gelatin  produces  similar 
colonies;  the  gelatin  is  liquefied  in  from  three  to  five  days.2  The 
colonies  are  rather  small  on  this  medium,  exhibit  radiating  edges,  and 
are  surrounded  by  a  liquefied  zone. 

Milk  is  both  coagulated  and  peptonized,  but  no  gas  is  formed  in  it. 

Blood  serum  is  rapidly  liquefied  and  it  is  an  excellent  medium  for 
the  development  of  this  organism. 

Artificial  cultures  possess  an  offensive  odor.  Broth  (anaerobic)  is 
clouded  by  the  organism  after  twelve  to  twenty-four  hours'  incubation, 
but  usually  clears  up  after  three  to  six  days.  The  organisms  grow 
much  better  in  albuminous  media,  particularly  those  containing  blood 
serum. 

Growth  does  not  take  place  below  15°  C.  nor  above  42°  C.  The 
optimum  temperature  is  37°  C.  The  organism  is  an  obligate  anaerobe 
and  sporulation  only  takes  place  anaerobically. 

1  Kutscher,  Ztschr.  f.  Hyg.,  1894,  xviii,  339.     Claudius,  Ann.  Inst.  Past.,  1897,  335- 

2  Liborius,  Ztschr.  f.  Hyg.,  1886,  i,  159, 


BACILLUS  (EDEMATIS  MALIGNI  495 

Resistance  to  Physical  Agents. — The  vegetative  cells  are  not  resistant 
to  heat,  three  to  five  minutes'  exposure  to  60°  C.  killing  them.  The 
spores  are  very  resistant  to  drying  and  heating;  an  exposure  to  80° 
C.  for  several  hours  is  necessary  to  kill  them,  and  from  thirty  to 
sixty  minutes'  exposure  to  90°  C.  Sunlight  will  not  kill  the  organisms 
even  after  several  days'  exposure,  and  in  the  dark  the  spores  may 
remain  alive  for  many  years.1 

Products  of  Growth. — B.  oedematis  maligni  forms  a  gelatinase,  case- 
ase,  and  apparently  a  non-specific  proteolytic  ferment  as  well.  The 
disagreeable  odor  noticed  in  protein  media  is  due  to  indol,  hydrogen 
sulphide  and  probably  mercaptans.  Acid,  chiefly  butyric  and  lactic, 
and  gas,  probably  carbon  dioxide  and  hydrogen,  are  produced  in 
dextrose  broth. 

Toxin. — It  has  been  claimed  that  the  bacillus  of  malignant  edema 
produces  a  soluble  toxin.  It  is  found  that  these  organisms  grown 
anaerobically  in  plain  broth  for  several  days  do  develop  a  slight 
toxicity,  which  can  be  demonstrated  by  filtering  the  broth  through 
sterile  unglazed  porcelain  filters  and  injecting  several  cubic  centi- 
meters of  the  filtrate  into  guinea-pigs;  they  die  after  a  longer  or 
shorter  time.  It  is  also  claimed  that  the  organism  produces  a  leuko- 
cidin  which  destroys  leukocytes. 

Pathogenesis. — The  virulence  of  cultures  of  the  malignant  edema 
bacillus  varies  very  considerably.  Infection  rarely  or  never  occurs  in 
man.  Brieger  and  Ehrlich2  have  reported  two  cases  of  typhoid  fever 
which  terminated  fatally  after  an  invasion  by  an  organism  morphologi- 
cally like  the  malignant  edema  bacillus,  which  produced  rather  exten- 
sive edema  in  the  tissues.  It  is  quite  possible  that  this  organism  was 
in  reality,  however,  the  gas  bacillus.  In  small  laboratory  animals, 
as  rabbits  and  guinea-pigs,  the  organism  typically  produces  a  rapidly 
fatal  septicemia  with  considerable  edema  at  the  site  of  injection. 
In  larger  animals,  horses,  cattle,  sheep  and  swine,  the  edema  is  more 
pronounced,  as  Koch3  pointed  out,  and  the  organism  tends  to  remain 
localized  at  the  site  of  inoculation.  As  a  rule  there  is  no  general  sep- 
ticemia. In  wound  infections  with  this  organism  the  incubation  period 
is  from  one  to  two  days.  Infection  only  takes  place  in  deep  or  con- 
tused wounds  where  oxygen  is  absent. 

Like  the  tetanus  bacillus,  the  spores  of  the  malignant  edema  bacilli, 
freed  from  adherent  culture  media  or  other  organisms,  do  not  as  a 

1  von  Sz&kely,  Ztschr.  f.  Hyg.,  1903,  xliv,  363. 

2  Berl.  klin.  Wchnschr.,  1882,  No.  44.  s  Loc.  cit. 


496  ANAEROBIC  BACTERIA 

rule  lead  to  infection.  The  spores  are  taken  up  by  phagocytes.  If 
the  spores  are  mixed  with  culture  filtrates,  with  weak  acids,  or  with 
other  organisms,  infection  usually  takes  place.  The  lesions  vary 
with  the  virulence  of  the  organism.  Organisms  of  moderate  virulence 
produce  edema  at  the  site  of  inoculation  which  tends  to  spread.  The 
regional  muscles  are  very  hyperemic  with  bubbles  of  gas  in  them, 
the  tissue  crepitates,  and  there  is  a  disagreeable  odor.  The  edema 
is  less  marked  and  death  takes  place  in  a  few  hours  when  organisms 
of  greater  virulence  are  injected.  Animals  appear  to  be  immune  after 
one  attack. 

Distribution. — The  organism  appears  to  be  very  widely  distributed 
in  the  soil  and  in  dust. 

Prophylaxis. — Prophylaxis  consists  essentially  in  immediate  surgical 
treatment  of  wounds  to  which  the  organism  might  gain  entrance. 

BACILLUS   ANTHRACIS   SYMPTOMATIC!. 

Historical. — The  disease  variously  known  as  black  leg,  quarter 
evil,  symptomatic  anthrax,  or  Rauschbrand  is  a  disease  of  cattle 
chiefly.  It  is  less  commonly  found  in  sheep  and  goats.  The  organism 
was  first  obtained  in  pure  culture  by  Arloing,  Cornevin,  and  Thomas.1 
The  organism  is  also  known  as  B.  chauvei  and  B.  sarcophysematis 
bo  vis. 

Morphology. — Morphologically,  it  is  a  rod-shaped  bacillus,  0.6  to 
1  micron  in  diameter  and  from  2  to  5  microns  in  length,  occurring 
singly  and  in  pairs.  It  practically  never  forms  chains,  differing  in 
this  respect  from  the  bacillus  of  malignant  edema.  The  organisms 
are  straight  and  rigid  and  have  square-cut  ends.  They  are  motile 
and  possess  many  peritrichic  flagella  and  form  no  capsules.  Spores 
occur  in  the  centre  of  the  organism  typically,  less  commonly  nearer 
one  end,  and  the  organism  is  slightly  swollen  because  the  spores  are 
slightly  greater  in  diameter  than  the  rod  itself.  It  stains  readily  with 
ordinary  anilin  dyes  and  is  Gram  positive. 

Isolation  and  Culture. — The  bacillus  of  symptomatic  anthrax  is  an 
obligate  anaerobe  which  grows  rather  poorly  in  artificial  media,  par- 
ticularly in  the  first  transfer  from  the  animal  body.  Albuminous 
media,  as  blood  serum,  or  blood  agar,  are  better  adapted  for  its  isola- 
tion than  ordinary  media.  It  grows  particularly  well  in  fermentation 
tubes  containing  sterile  tissue,  according  to  Theobald  Smith's  method. 

1  Le  Charbon,  Symptomatique  du  Boeuf,  Paris,  1887. 


BACILLUS  ANTHRACIS  SYMPTOMATICI  497 

Material  for  inoculation  is  best  obtained  from  the  heart's  blood,  the 
local  swelling,  or  the  peritoneal  exudate  of  an  animal  dead  of  the 
disease.  The  material  should  be  sown  anaerobically  on  ascitic  or  blood 
agar  plates  or  upon  dextrose  agar,  the  latter  medium  not  being  as 
satisfactory.  Pure  cultures  may  be  obtained  readily  by  inoculating 
guinea-pigs  with  morbid  material  and  transferring  some  of  the  heart's 
blood  of  the  animal  immediately  after  death  to  artificial  media. 

Growth  on  Artificial  Media. — The  organism  grows  to  a  limited  extent 
in  plain  broth  if  oxygen  is  excluded.  It  grows  better  in  dextrose 
broth.  On  anaerobic  dextrose  gelatin  and  dextrose  agar  plates  the 
colonies  are  round,  oval,  grayish,  and  possess  distinctly  filamentous 


FIG.  70. — Bacillus  of  symptomatic  anthrax  spore  formation.     X  1000. 

edges.  Gelatin  is  liquefied  in  from  two  to  four  days.  Milk  is  a  good 
medium;  the  organism  forms  a  slight  amount  of  acid,  but  no  coagula- 
tion or  peptonization  takes  place. 

Conditions  of  Growth. — B.  anthracis  symptomatici  is  an  obligate 
anaerobe  which  does  not  grow  below  14°  C.  nor  above  44°  C.  The 
optimum  temperature  is  37°  C.  The  spores  are  extremely  resistant  to 
heat;  half  an  hour's  exposure  to  100°  C.  does  not  always  kill  them. 
The  spores  appear  to  be  able  to  remain  latent  in  the  animal  body. 
The  virulence  of  vegetative  organisms  developing  from  spores  is  said 
to  be  greatly  reduced  by  heating  the  spores  to  100°  C.  for  two  to  three 
minutes. 

Products  of  Growth. — The  organism  forms  a  gelatinase.  In  dextrose 
broth  it  produces  carbon  dioxide,  hydrogen,  and  traces  of  methane, 
as  well  as  butyric  and  lactic  acids. 

32 


498  ANAEROBIC  BACTERIA 

Toxin. — According  to  Leclainche  and  Vallee,1  and  Grassberger  and 
Schattenfroh,2  the  filtrates  of  broth  cultures  of  the  bacillus  are  slightly 
toxic  to  guinea-pigs  in  large  doses. 

Pathogenesis. — The  organism  is  not,  so  far  as  is  known,  pathogenic 
for  man.  At  the  site  of  inoculation  in  animals  there  is  a  rapidly 
spreading  edema  which  appears  to  be  very  painful.  Usually  the 
most  prominent  naturally  occurring  lesion  is  a  swelling  of  the  front 
or  hind  quarters;  the  lesion  practically  never  extends  below  the  knee. 
The  edematous  area  is  almost  black,  due  apparently,  in  part  at  least, 
to  changed  blood  pigment,  and  the  area  is  surrounded  by  a  zone  of 
hyperemia.  The  hair  over  the  edematous  area  falls  out  easily.  There 
is  considerable  degeneration  of  the  muscular  tissue  in  the  edematous 
zone,  and  there  is  in  it  a  sanguineous  exudate  which  contains  relatively 
few  leukocytes.  The  edematous  area  is  crepitant,  due  to  accumulated 
gas  bubbles,  and  there  is  a  rather  strong  odor  of  butyric  acid.  The 
incubation  period  is  from  one  to  three  days. 

Sporulation  does  not  take  place  in  the  tissues  of  the  living  animal, 
but  it  is  said  to  take  place  in  from  twenty-four  to  forty-eight  hours 
after  death.  If  the  spores  are  washed  free  from  toxin  and  other  bac- 
teria they  are  said  not  to  be  infective  for  experimental  animals, 
according  to  Leclainche  and  Vallee.3 

Vaccine. — One  attack  of  symptomatic  anthrax  appears  to  confer 
immunity  to  subsequent  attacks.  Young  cattle  are  usually  infected; 
older  ones  appear  to  be  more  resistant  to  infection.  A  vaccine  has 
been  prepared  which  protects  the  animal  from  infection.  The  general 
process  of  manufacture  is  to  remove  the  infected  tissues  of  animals 
dead  of  symptomatic  anthrax  and  dry  them  under  aseptic  conditions 
at  37°  C.4  From  this  dried  tissue  two  vaccines  are  made  up,  the  first 
being  prepared  by  mixing  the  dried  powder  with  sterile  water5  to  form 
a  paste,  which  is  heated  to  100°  C.  for  six  hours.  This  is  the  first 
vaccine,  which  will  not  kill  experimental  animals.  It  is  injected  at 
the  tip  of  the  tail.  In  seven  days  a  second  vaccine  (prepared  from 
the  same  powder  and  heated  to  94°  C.  for  four  hours)  is  injected  in 
the  same  manner.  This  vaccine  will  ordinarily  kill  small  experimental 
animals.  These  two  vaccines  or  modifications  of  them  are  widely 
used  for  protecting  cattle  against  blackleg. 

1  Ann.  Inst.  Past.,  1900,  202. 

2  Uber  das  Rauschbrandgift,  1904.  3  Loc.  cit. 

4  This  temperature  does  not  diminish  the  virulence  of  the  bacteria ;    the  potency  of 
the  dried  virus  remains  unimpaired  for  eighteen  to  twenty-four  months. 
6  Two  parts  sterile  water  to  one  part  of  dried  powder. 


,      CHAPTER  XXVI. 


THE  CHOLERA  GROUP. 


CHOLERA  VIBRIO. 


Vibrio  of   Finkler  and   Prior    (Vibrio 
Proteus). 


Vibrio  Metchnikovi. 

Vibrio  Massaua. 

Vibrio  Tyrogenum  (Spirillum  Deneke). 


MANY  vibrios  have  been  described  which  possess  in  common  with 
the  cholera  vibrio  a  number  of  cultural  characters.  They  are  all 
comma-shaped  organisms,  Gram-negative,  possess  a  terminal  flagellum, 
form  no  spores  or  capsules,  and  liquefy  gelatin  more  or  less  rapidly. 
They  dift'er  among  themselves  culturally  chiefly  with  respect  to  the 
intensity  with  which  these  reactions  occur.  Some  produce  nitroso 
indol  in  sugar-free  culture  media,  others  produce  indol  only.  They 
may  be  sharply  differentiated  from  the  true  cholera  vibrio  by  serum 
reactions.  So  far  as  is  known,  none  of  these  organisms  will  agglutinate 
with  a  specific  cholera  immune  serum  in  high  dilution,  1  to  2000  to 
1  to  5000,  depending  upon  the  titre.  None  of  these  organisms  are 
dissolved  by  cholera  immune  serum  (Pfeiffer  reaction).  The  true 
cholera  vibrio  gives  these  serum  reactions.  Most  of  these  organisms 
have  been  isolated  from  water.  Even  within  the  group  of  the  true 
cholera  cultures,  that  is,  those  which  react  with  a  specific  cholera 
immune  serum,  there  appear  to  be  varieties  which  are  distinguishable 
from  the  type  organism  with  great  difficulty.  The  principal  variants 
are  described  below. 

CHOLERA   VIBRIO. 

Synonyms. — Vibrio  cholerse  asiaticae,  Spirillum  choleras  asiaticae, 
comma  bacillus,  cholera  vibrio. 

Historical. — The  cholera  vibrio  was  first  isolated  in  pure  culture 
by  Koch  in  1883.1  For  some  years  the  organism  was  not  universally 
accepted  as  the  causative  agent  in  Asiatic  cholera,  and  some  weight 
was  attached  to  the  frequent  isolation  of  vibrios  very  similar  in  mor- 
phological and  cultural  characters  to  the  true  cholera  vibrio  from  the 
dejecta  of  normal  individuals.  These  cholera-like  vibrios  were  not 

1  Deutsch.  med.  Wchnschr.,  1883,  615,  743;  1884,  63,  111,  221,  499,  519;  1885,  No. 
37a;  British  Med.  Jour.,  1884,  ii,  403,  453. 


500  THE  CHOLERA   GROUP 

sharply  differentiated  from  the  true  cholera  vibrio  with  the  imperfect 
methods  available  in  the  early  days  of  bacteriology,  when  these  obser- 
vations were  made.  It  is  now  universally  held  that  the  cholera  vibrio 
is  the  causative  organism  of  the  disease. 

Morphology. — The  typical  cholera  vibrio  is  a  distinctly  curved  rod, 
the  curvature  being  in  three  planes  of  space.  It  measures  0.5  to  0.6 
micron  in  diameter  by  1  to  3  microns  in  length,  occurring  singly  or 
in  pairs,  less  commonly  in  longer  spiral  chains  of  several  elements. 
Pairs  of  organisms  frequently  appear  as  S-shaped  spirilla,  the  curva- 
ture being  in  three  planes  of  space  in  the  living  vibrios.  Freshly 
isolated  vibrios  have  slightly  but  distinctly  pointed  ends  which  are 


FIG.  71. — Cholera  vibrios,  showing  flagella. 

best  observed  in  stained  specimens  made  directly  from  cholera  dejecta. 
Cultures  grown  for  some  time  on  artificial  media  lose  their  original 
uniformity  of  size  and  shape  and  tend  to  become  less  curved,  many 
individuals  even  appearing  as  straight  rods.  The  passage  of  these  old 
cultures  through  animals  is  said  to  restore  their  original  morphology. 
Cultures  in  artificial  media  several  days  old  frequently  exhibit  involu- 
tion forms  which  are  irregularly  swollen  or  even  coccoid  in  outline. 
Bacillary  forms  and  even  true  spirillum  forms  also  are  not  uncommonly 
seen. 

Cholera  vibrios  are  actively  motile  and  they  possess  a  single  polar 
flagellum — monotrichic  flagellation.1  No  capsule  has  been  demon- 
strated and  no  spores  are  produced,  although  involution  forms  which 
stain  somewhat  irregularly  may  suggest  spores. 

The  cholera  organism  stains  with  ordinary  anilin  dyes,  although  less 

1  Loffler,  Centralbl.  f.  Bakt.,  1889,  vi>  209. 


CHOLERA   VIBRIO  501 

readily  than  the  majority  of  pathogenic  bacteria.  This  is  particularly 
the  case  in  freshly  isolated  cultures.  Older  cultures  are  more  uniform 
in  this  respect.  The  organism  is  invariably  Gram-negative. 

Isolation  and  Culture. — Cholera  vibrios  grow  rapidly  upon  all  ordinary 
artificial  media,  even  at  20°  C.  Their  nutritional  requirements  with 
respect  to  nitrogenous  substances  are  less  exacting  than  those  of 
many  pathogenic  and  non-pathogenic  bacteria  commonly  found  in 
the  intestinal  tract.  Also  the  true  cholera  organisms  are  tolerant  of 
a  degree  of  alkalinity  which  is  unsuited  for  the  development  of  ordinary 
bacteria.  Advantage  is  taken  of  these  nutritional  peculiarities  in 
isolating  cholera  vibrios  from  the  dejecta  of  cholera  patients.  A 
small  portion  of  fecal  mucus  is  emulsified  in  slightly  alkaline  Dunham's 
solution1  and  incubated  for  six  to  eight  hours  at  37°  C.  The  cholera 
organisms  increase  in  numbers  with  great  rapidity  and  they  will  be 
found  at  the  surface  of  the  medium  in  considerable  concentration, 
for  they  are  strongly  aerobic.  The  isolation  of  them  in  pure  culture 
by  plating  is  readily  accomplished  if  the  material  for  inoculation  is 
taken  from  the  surface  of  such  a  peptone  culture.2 

Growth  in  Artificial  Media. — Colonies  of  cholera  vibrios  which  appear 
on  agar  plates  after  twelve  to  eighteen  hours'  incubation  at  37°  C. 
are  round,  very  thin  and  transparent,  and  when  viewed  by  transmitted 
light  they  are  nearly  colorless.  Colonies  of  colon  and  other  intestinal 
bacteria  are  usually  yellowish  brown  under  the  same  conditions. 
The  colonies  of  freshly  isolated  cholera  vibrios  are  even  more  trans- 
parent than  colonies  of  typhoid,  paratyphoid,  or  dysentery  bacilli. 
Older  cultures  do  not  exhibit  this  transparency  to  such  a  degree. 

Colonies  on  gelatin  plates  present  a  somewhat  characteristic 
appearance.  After  twenty  to  twenty-four  hours'  incubation  the 
organisms  have  produced  a  slight  liquefaction  which  gives  the  surface 
of  the  medium  a  "ground-glass"  appearance  when  the  plate  is  viewed 
at  an  acute  angle.  Liquefaction  proceeds  rapidly.  The  cultures  which 
have  been  grown  on  artificial  media  for  a  long  time  liquefy  gelatin 
more  slowly  and  eventually  may  lose  this  property.  In  sugar-free 
gelatin  stab  cultures  an  "air  bubble,"  so  called,  frequently  forms 
just  below  the  surface  of  the  medium.  This  probably  is  the  result  of 
the  evaporation  of  water  from  the  liquefied  medium.  No  liquefaction 
takes  place  in  sugar  gelatin. 

1  Dunham  solution:    Peptone  1  gram,  NaCl  0.5  gram,  potassium  nitrate  0.25  gram, 
sodium  carbonate  (cryst.)  0.5  gram,  water  100  c.c. 

2  See  Bacteriological  diagnosis  for  details. 


502  THE  CHOLERA   GROUP 

Blood  serum  is  liquefied.  Broth  is  densely  clouded  and  in  plain 
broth  or  in  Dunham's  solution  a  pellicle  is  usually  formed  after  twelve 
to  twenty-four  hours'  growth.  A  pellicle  does  not  ordinarily  develop 
in  sugar-containing  broth. 

Milk  is  acidified,  the  degree  of  acid  produced  varying  greatly  with 
the  strain  of  organism.  Some  cultures  produce  enough  acid  to  cause 
acid  coagulation  of  the  milk.  No  peptonization  takes  place.  Litmus 
milk  is  not  coagulated. 

The  production  of  hemolysis  (erythrocytolysis)  by  cholera  vibrios 
is  a  subject  of  controversy.  It  was  formerly  maintained  that  vibrios 
which  agglutinate  at  high  dilution  with  specific  cholera  sera  of  high 


FIG.  72. — Cholera  vibrios  from  feces. 

potency  were  non-hemolytic.  The  consensus  of  opinion  at  the  present 
time  concedes  that  a  moderate  proportion  of  typical  cholera  vibrios 
are  hemolytic,  although  the  active  hemolysin  can  not  always  be 
obtained  in  a  soluble  form.  This  property  is  shared  by  many  cholera- 
like  organisms.  A  group  of  vibrios,  of  which  two  strains,  Vibrio  Nasik, 
and  Vibrio  El  Tor,  are  the  best  known,  are  so  closely  related  to  the 
cholera  vibrio  that  they  have  caused  much  study  and  speculation. 
The  former  fails  to  agglutinate  with  a  specific  cholera  serum,  but  is 
strongly  hemolytic;  the  latter  also  fails  to  agglutinate  at  high  dilution, 
although  it  acts  as  an  antigen  with  cholera  serum  in  the  complement- 
fixation  test.  It  produces  a  thermostabile  soluble  toxin.1 

The  organisms  are  aerobic,  facultatively  anaerobic.  They  were 
formerly  considered  to  be  strongly  aerobic;  it  is  doubtful,  however, 
if  they  are  markedly  more  aerobic  than  other  intestinal  bacteria.  The 

1  See  Kraus  and  Pribram,  Wien.  klin.  Wchnschr.,  1905,  No.  39. 


CHOLERA   VIBRIO  503 

limits  of  growth  are  10°  C.  and  43  to  45°  C.  respectively,  the  optimum 
being  37°  C.  They  are  very  sensitive  to  drying;  according  to  Giinther,1 
three  hours'  drying  kills  them.  They  remain  alive,  however,  for  weeks 
in  culture  media.  An  exposure  to  60°  C.  for  thirty  minutes  usually 
kills  them.  Freezing  at  10°  C.  has  little  effect  even  if  the  exposure  is 
prolonged.  They  will  remain  viable  in  impure  water  for  from  one  to 
two  weeks  on  the  average. 

In  feces  they  may  remain  alive  for  seven  to  nine  months  if  air  is 
excluded,  according  to  Zlatogoroff.2  Under  ordinary  conditions, 
however,  they  remain  viable  for  much  shorter  periods  of  time  in  feces. 
According  to  Forster,3  the  organisms  are  very  sensitive  to  acids 
and  to  germicides.  According  to  his  observations,  a  dilution  of  1 
to  300,000  bichloride  of  mercury  kills  them  in  five  minutes,  and 
1  to  3,000,000  in  ten  minutes.  These  results  have  not  been  corrob- 
orated and  it  is  very  likely  that  they  are  not  markedly  more  sensitive 
to  disinfectants  than  the  ordinary  pathogenic  intestinal  bacteria,  as 
the  typhoid  bacillus.  Behring4  has  found  that  0.5  per  cent,  carbolic 
acid  will  nearly  kill  cholera  organisms  after  an  exposure  of  an  hour. 
Bichloride  of  mercury  in  a  dilution  of  1  to  1000  kills  them  in  ten 
minutes,  and  5  per  cent,  carbolic  in  less  than  fifteen  minutes. 

Products  of  Growth.  —  Cholera  organisms  produce  in  sugar-free  pro- 
tein media  an  active  soluble  gelatinase  which  dissolves  gelatin  and  also 
blood  serum.  Some  strains  elaborate  a  soluble  hemolysin.5  No  other 
enzymes  are  known. 

One  of  the  striking  reactions  of  the  organism  is  the  so-called 
"cholera-red  reaction,"  or  the  nitroso  indol  reaction.  The  addition 
of  acid,  either  sulphuric  or  hydrochloric  or  nitric,  to  a  forty-eight-hour 
culture  of  cholera  vibrios  grown  in  sugar-free  nutrient  broth  or  in 
peptone  solution,  will  develop  the  well-known  reddish-brown  color 
indicative  of  the  indol  reaction.  The  organisms  appear  to  form 
nitrites  from  the  protein  constituents  of  the  medium.  The  reactive 
substance  was  regarded  by  Poehl6  as  a  skatol  derivative.  This  view 
appears  to  have  been  accepted  by  Bujwid7  and  Dunham.8  Brieger,9 
however,  regards  it  as  an  indol  derivative.  It  is  probable  that  Brieger's 
explanation  is  the  correct  one.  The  substance  formed  is  nitroso  indol, 


1  Bakteriologie,  p.  644.  2  Centralbl.  f.  Bakt.,  1911,  Iviii,  14. 

3  Hyg.  Rund.,  1893,  722.  *  Ztschr.  f.  Hyg.,  1890,  ix,  400. 

6  See  Public  Health  Reports,  1912,  xxvii,  No.  11,  for  full  details. 
6Ber.  d.  deutsch.  chem.  Gesell.,  1886,  xix,  1162. 

7  Centralbl.  f.  Bakt.,  1888,  iv,  494. 

8  Ztschr.  f.  Hyg.,  1887,  ii,  340.  9  Deutsch.  med.  Wchnschr.,  1887,  No.  15. 


504  THE  CHOLERA   GROUP 

the  indol  radical  being  derived  from  the  decomposition  of  tryptophan. 
The  same  reaction  may  be  obtained  from  the  rice  water  stools  of 
cholera  patients.  The  cholera-red  reaction  is  not  produced  in  media 
containing  utilizable  carbohydrates.1  The  nitroso  indol  or  cholera-red 
reaction  is  not  specific  for  the  cholera  vibrio.  Other  closely  related 
bacteria  also  give  the  same  reaction.  On  the  other  hand,  not  all  true 
cholera  vibrios  form  nitroso  indol. 

Besides  nitroso  indol,  cholera  vibrios  produce  considerable  amounts 
of  ammonia  and  hydrogen  sulphide  in  sugar-free  media.2  All  true 
cholera  vibrios  produce  acid  in  dextrose  and  lactose.  The  production 
of  acid  in  saccharose  and  mannite  is  somewhat  less  constant.  The 
acids  produced  are  levolactic  acid,3  also  acetic  and  butyric  acids.4 

Toxin. — The  nature  of  the  poison  or  poisons  produced  by  the  cholera 
vibrio  is  still  a  subject  of  controversy,  although  the  disease  cholera 
appears  to  be  a  toxemia,  for  the  organisms  do  not  commonly  invade 
the  tissues  of  the  body  even  in  fatal  cases.  Pfeiffer's  view5  was  that 
the  toxin  is  an  endotoxin  which  is  liberated  by  autolysis  from  the 
organisms  themselves.  Behring  and  Ransom,6  on  the  contrary, 
claim  to  have  separated  a  soluble  toxin  from  broth  cultures  of  true 
cholera  vibrios  which  in  doses  of  about  0.5  c.c.  will  kill  guinea-pigs  in 
twenty-four  hours.  They  further  claim  to  have  immunized  guinea- 
pigs  and  goats  to  the  toxin  by  injecting  gradually  increasing  doses. 
The  antitoxin  thus  obtained  protects  non-immune  animals  against 
the  toxin  or  from  infection  with  living  cholera  vibrios.  The  toxin  is 
unaffected  by  moderate  heat,  chloroform,  toluol,  or  carbolic  acid. 

Metchnikoff  Roux  and  Taurelli-Salimbini7  enclosed  peptone  cul- 
tures of  cholera  vibrios  in  collodion  sacs  which  were  placed  in  the 
peritoneal  cavities  of  guinea-pigs.  As  controls,  killed  cultures  of 
cholera  vibrios  and  sterile  uninoculated  peptone  respectively  were 
placed  in  other  guinea-pigs  in  collodion  sacs.  The  guinea-pigs  which 
received  only  sterile  peptone  solution  in  capsules  failed  to  show 
symptoms;  those  containing  killed  cultures  of  cholera  vibrios  in 
capsules  showed  a  slight  febrile  reaction  and  some  emaciation;  the 
guinea-pigs  which  received  the  collodion  capsules  containing  living 
cholera  vibrios  died  after  three  to  five  days  with  symptoms  of  choleraic 

1  Gorini,  Centralbl.  f.  Bakt.,  1893,  xiii,  790.     Kendall,  Boston  Med.  and  Surg.  Jour., 
1913,  clxviii,  825. 

2  Kendall,  Day  and  Walker,  Jour.  Biol.  Chem.,  1913,  xxxv,  1240. 
8  Kuprianow,  Arch.  f.  Hyg.,  1893,  xix,  288. 

«Gosio,  Arch.  f.  Hyg.,  1894,  xxi,  120;    1894,  xxii,  11. 

5  Centralbl.  f.  Bakt.,  Ref.,  1892,  xi,  568.  "Ibid.,  1895,  xviii,  314. 

7  Ibid.,  1896,  xx,  627. 


CHOLERA    VIBRIO  505 

intoxication.  These  observers  concluded  from  these  experiments 
that  the  cholera  organism  produced  a  soluble  toxin  which  was  dif- 
fusible through  collodion  sacs.  The  toxicity  of  these  cultures  was  not 
destroyed  by  the  boiling  temperature,  100°  C.  They  were  able  to 
immunize  guinea-pigs,  rabbits,  goats,  and  horses  with  this  so-called 
soluble  toxin,  and  found  the  serum  of  these  animals  was  antitoxic  and 
protective  against  several  times  the  fatal  dose  of  toxin  or  of  the  living 
organisms.  Antitoxic  sera  prepared  by  this  method  have  not  been 
successful  in  the  clinical  treatment  of  cholera  in  man.  It  is  not  unlikely 
that  the  soluble  toxic  substance  or  substances  produced  in  artificial 
cultivations  of  the  cholera  vibrio  play  a  less  important  part  in  the 
disease  than  the  endotoxins,  which  appear  to  be  liberated  from  the 
organism  with  unusual  readiness. 

The  extremely  brief  period  which  elapses  between  infection  and 
death,  twelve  hours  in  unusual  cases,  would  suggest  that  the  incubation 
period  of  the  cholera  toxin,  if  such  play  a  part  in  the  disease,  is  very 
much  less  than  that  of  any  other  known  soluble  bacterial  toxin. 

Pathogenesis. — Animal. — Different  strains  of  cholera  vibrios  vary 
greatly  in  their  virulence  for  experimental  animals;  prolonged  cultiva- 
tion on  artificial  media  tends  to  diminish  their  pathogenicity  as  a 
rule.  Virulent  cultures  injected  intraperitoneally  in  experimental 
animals,  particularly  guinea-pigs,  frequently  cause  acute  peritonitis; 
the  animal  gradually  sinks  into  a  state  of  coma,  the  temperature  falls, 
and  death  intervenes  with  or  without  convulsions.  At  autopsy  the 
peritoneum  is  reddened,  the  peritoneal  surface  of  the  intestines  is 
greatly  congested,  and  there  are  usually  small  ecchymoses.  There 
is  some  increase  in  the  peritoneal  fluid,  which  frequently  contains 
vibrios.  They  may  also  be  found  in  the  blood  stream  as  well.  Sub- 
cutaneous injections  of  like  amounts  of  culture  may  or  may  not 
result  fatally.  The  organisms,  however,  as  Theobald  Smith  pointed 
out  many  years  ago,  tend  to  migrate  to  the  intestinal  tract,  suggesting 
that  some  chemotactic  influence  attracts  them  there.  Intravenous 
injection,  particularly  in  young  rabbits,  may  lead  to  lesions  in  the 
intestinal  tract,  suggesting  those  characteristic  of  cholera  in  man, 
but  as  a  rule  far  less  severe.  The  organisms  may  also  be  found  in  the 
intestinal  contents  and  gall-bladder  following  intravenous  injection. 

Feeding  experiments  in  the  ordinary  way  are  not  successful.  Koch1 
succeeded  in  infecting  young  guinea-pigs  with  cholera  vibrios  by  first 

1  Deutsch.  med.  Wchnschr.,  1885,  No.  37a,  5-6. 


506  THE  CHOLERA  GROUP 

administering  sodium  carbonate  by  mouth  to  neutralize  the  gastric 
acidity,  then  introducing  by  mouth  10  c.c.  of  a  broth  culture  of  the 
vibrios  directly  into  the  stomach  with  a  catheter.  The  animals  died 
usually  in  about  two  days  with  symptoms,  and  particularly  intestinal 
lesions  which  resembled  those  of  cholera  in  man.  There  were  diarrhea, 
bloody  rice-water  stools  with  abundant  organisms  in  them,  collapse 
and  death.  Issaeff  and  Kolle1  made  similar  experiments  in  young 
rabbits,  and  Wiener2  has  successfully  infected  kittens  in  the  same 
way. 

Human. — (a)  Experimental  Evidence  of  Disease. — In  man  infection 
takes  place  usually  by  ingestion  of  food  or  water  contaminated  with 
cholera  vibrios.  The  first  accidental  laboratory  infection  is  probably 
that  mentioned  by  Koch3  of  a  doctor  who  accidentally  swallowed  part 
of  a  culture  and  contracted  the  disease.  Hasterlik,4  Metchnikoff,5 
Reners,6  Kolle7  and  Voges8  have  also  reported  laboratory  infections 
of  man  with  cholera  vibrios  which  resulted  in  typical  disease  in  each 
instance,  thus  establishing  beyond  reasonable  doubt  the  etiological 
relation  of  the  cholera  vibrio  to  the  disease  cholera. 

(6)  Natural  Infection. — The  incubation  period  of  the  naturally 
acquired  disease  cholera  may  be  very  short;  the  patient  may  be 
infected  and  die  within  twelve  hours,  so-called  cholera  sicca.9  Ordi- 
narily the  incubation  period  is  from  one  to  two  days.10 

The  important  clinical  symptoms  are  extremely  painful  cramps, 
great  withdrawal  of  water  from  the  tissues,  due  to  the  violent  diarrhea, 
resulting  in  shriveling  of  the  skin  of  the  extremities  and  increased 
viscosity  of  the  blood.  The  urine  after  the  first  day  is  scanty  in 
amount,  the  stools  are  very  fluid,  "rice-water  stools,"  and  there  is 
profound  collapse.  The  most  noteworthy  lesions  postmortem  are 
in  the  small  intestine,  particularly  the  lower  half.  The  mucosa  is 
swollen  and  congested  particularly  about  Peyer's  patches;  the  contents 
of  the  intestinal  tract  are  fluid  and  contain  shreds  of  mucus.  There 
is  parenchymatous  degeneration  of  the  liver,  kidneys  and  spleen. 
The  intestinal  contents  swarm  with  vibrios.  In  the  markedly  chronic 
cases  there  may  be  extensive  necrosis  and  serofibrinous  exudation  on 
the  surface  of  the  intestinal  mucosa. 


1  Ztschr.  f.  Hyg.,  1894,  xviii,  17.  2  Centralbl.  f.  Bakt.,  1896,  xix,  205. 

3  Deutsch.  med.  Wchnschr.,  1885,  No.  37a,  7.     4  Wien.  klin.  Wchnschr.,  1893,  167. 
5  Ann.  Inst.  Past.,  1893,  No.  7.  6  Deutsch.  med.  Wchnschr.,  1894,  52. 

'  Ztschr.  f.  Hyg.,  1894,  xviii,  17.  8  Centralbl.  f.  Bakt.,  1895,  xviii,  629. 

9  Metchnikoff,  Ann.  Inst.  Past.,  1893,  581. 
10  Banti,  Lo  Sperimentale,  1887.     Gunther,  Deutsch.  med.  Wchnschr.,  1892,  841. 


CHOLERA   VIBRIO  507 

Immunity. — As  a  rule  one  attack  confers  lasting  immunity. 

Artificial  Immunity. — Attempts  have  been  made  to  induce  artificial 
active  immunity: 

1.  By  subcutaneous  inoculation  of  virulent  cholera  vibrios  in  man, 
either  directly  or  after  exaltation  of  their  virulence  for  guinea-pigs 
or  rabbits.1  (2)  By  the  injection  of  autolyzed  cultures  of  cholera 
vibrios,  heated  at  60°  C.  for  an  hour  to  kill  them,  then  suspended  in 
distilled  water  at  37°  C.  for  three  to  four  days,  and  filtered  through 
porcelain.2  (3)  Vaccines,  (a)  Killed  cultures  (Kolle);  (b)  sensitized 
cultures  (Besredka);  (c)  bacterial  extractives. 

The  only  method  thus  far  which  has  yielded  encouraging  results 
is  that  of  HarTkine.3  This  consists  in  the  injection  of  from  0.25  to  0.5 
c.c.  of  a  suspension  of  an  agar  culture  of  cholera  vibrios  suspended 
in  5  c.c.  of  sterile  saline  solution.  This  is  introduced  subcutaneously. 
The  results  reported  from  India  are  claimed  to  be  favorable. 

Bacteriological  Diagnosis. — Isolation  and  identification  of  the  cholera 
vibrio. 

1.  Microscopic. — The  feces  may  be  examined  directly  for  cholera 
vibrios.    Large  numbers  of  slightly  curved  or  S-shaped  actively  motile 
vibrios,  which  when  stained  with  dilute  carbolfuchsin  exhibit  slightly 
tapered  ends,  are  very  suggestive.    A  bit  of  mucus  (a  "grain  of  rice" 
from  a  rice-water  stool)  is  particularly  good  for  microscopical  exam- 
ination.    The  organisms  frequently  exhibit  a  marked  parallelism  of 
their  long  axes,  resembling  a  school  of  fish  in  their  arrangement  if 
the  material  is  not  roughly  handled  during  the  preparation  of  the 
smear. 

2.  Culture. — (a)    Schottelius'    method.      The   principle    involved: 
The  cholera  vibrio  grows  particularly  well  in  alkaline  peptone  solu- 
tion (Dunham  solution).    Bacillus  coli  and  other  intestinal  organisms 
grow  less  readily. 

Technic. — A  loopful  of  feces,4  or  preferably  a  small  piece  of  mucus 
is  emulsified  in  a  tube  of  Dunham's  peptone  solution  and  incubated 
at  37°  C.  for  six  to  eight  hours.  The  cholera  organisms  are  very 
aerobic  and  actively  motile,  and  collect  in  large  numbers  at  the 
surface  of  the  medium,  therefore  two  or  three  loopfuls  of  material 
from  the  surface  of  the  Dunham  tube  are  inoculated  into  a  second 

1  Haffkine.  2  Haffkine  and  Ferran.  3  Bull.  Inst.  Past.,  iv,  697,  737. 

4  If  the  preliminary  microscopical  examination  fails  to  reveal  a  preponderance  of 
vibrios  of  characteristic  morphology,  a  larger  amount  of  fecal  material  must  be  taken. 
Several  grams  of  feces  emulsified  in  100  to  500  c.c.  Dunham  solution  may  give  positive 
results  in  exceptional  cases  when  smaller  samples  are  negative. 


508  THE  CHOLERA  GROUP 

tube  and  the  process  repeated  in  a  third  tube,  when  a  nearly  pure 
culture  of  cholera  vibrios  will  frequently  be  obtained.  The  organisms 
may  be  plated  directly  from  the  enriched  growth  in  the  first,  second, 
or,  best,  from  the  third  tube,  and  the  pure  cultures  agglutinated  with 
a  high  potency  specific  anticholera  serum  in  dilutions  from  1  to  500 
to  1  to  5000. l  The  nitroso  indol  test  should  be  made  on  each  of  the 
three  Dunham  tubes  after  removal  of  the  organisms,  for  a  positive 
nitroso  indol  reaction,  while  not  diagnostic,  is  very  suggestive. 

(6)  A  small  amount  of  feces  or  a  flake  of  mucus  is  emulsified  in 
broth  and  inoculated  on  the  surface  of  alkaline  agar  plates,2  which 
have  previously  been  poured  and  hardened.  The  very  thin  trans- 
parent colonies  which  develop  within  twelve  to  eighteen  hours  are 
either  transferred  to  broth  and  after  twelve  hours'  incubation  agglu- 
tinated, or  the  colony  is  emulsified  directly  in  a  high  potency  specific 
serum  diluted  five  hundred  times  and  a  macroscopic  or  microscopic 
examination  made.  Controls  are  made  using  either  normal  serum 
diluted  twenty-five  times,  or  normal  salt  solution.  Cholera  vibrios 
will  agglutinate  rapidly  while  the  controls  remain  actively  motile. 

3.  Agglutination  of  Organism. — (a)  A  pure  culture  of  cholera  vibrios 
will  agglutinate  in  high  dilutions  with  a  high  potency  specific  cholera 
serum  either  by  the  microscopic  or  macroscopic  agglutination  method. 
The  macroscopic  agglutination  test  can  be  made  either  by  preparing 
successive  dilutions  of  the  antiserum  in  small  tubes,  1  to  250  up  to 
1  to  2500,  and  adding  an  equal  volume  of  broth  culture  of  cholera 
vibrios  to  each,  or  by  making  dilutions  of  the  specific  serum  1  to  500 
up  to  1  to  5000  in  small  tubes  and  emulsifying  in  each  tube  a  smaK 
amount  of  culture  from  an  agar  slant.  Appropriate  controls  should 
be  made  in  either  case.  A  positive  diagnosis  of  cholera  vibrios  should 
only  be  made  if  agglutination  takes  place  with  a  specific  anticholera 
serum  in  a  dilution  of  at  least  1  to  500. 

(6)  A  flake  of  mucus  containing  many  vibrios  is  emulsified  directly 
in  specific  anticholera  serum,  diluted  at  least  1  to  500,  and  a  suitable 
control  is  made  with  normal  serum.  This  is  best  carried  out  by  the 
microscopic  agglutination  method.  A  positive  agglutination  under 

1  The  anticholera  serum  "is  best  obtained  from  rabbits  which  have  been  immunized 
by  repeated  injections  of  known  cholera  vibrios.     The  titre  of  the  serum  should  be  at 
least  1  to  4000.     A  final  diagnosis  should  be  made  preferably  only  when  the  suspected 
organism  agglutinates  in  a  dilution  of  at  least  1  to  2000,  although  clumping  of  freshly 
isolated  vibrios  at  a  dilution  of  1  to  500  is  fairly  conclusive.     The  sera  of  horses  and 
other  large  animals  are  less  suitable  for  agglutination  with  cholera  vibrios;     natural 
antibodies  occur  which  cause  clumping  in  relatively  high  dilutions. 

2  The  necessary  degree  of  alkalinity  may  be  attained  by  adding  3  c.c.  of  a  10  per  cent, 
solution  of  sodium  carbonate  to  each  100  c.c.  of  neutral  (litmus)  agar. 


CHOLERA    VIBRIO  509 

these  conditions  is  fairly  conclusive.  It  should  be  remembered  that 
an  occasional  strain  of  the  cholera  vibrio  is  met  with  which  does  not 
agglutinate  when  freshly  isolated;  prolonged  cultivation  in  artificial 
media  frequently  leads  to  a  typical  agglutination. 

4.  Identification  of  Cholera  Vibrios  by  the  Pfeiffer  Phenomenon. — 
If  cholera  vibrios  are  introduced  directly  into  the  peritoneal  cavity 
of  an  immunized  guinea-pig  and  samples  of  the  peritoneal  exudate 
containing  vibrios  are  removed  from  the  peritoneal  cavity  with  a 
capillary  pipette  after  ten  minutes,  sixty  minutes  and  ninety  minutes, 
it  will  be  found  that  usually  after  ten  minutes,  almost  invariably 
within  an  hour,  the  vibrios  will  become  very  much  granulated  and  will 
eventually  dissolve.  A  normal  guinea-pig  similarly  infected  intra- 
peritoneally  with  a  mixture  of  cholera  vibrios  and  immune  serum 
will  exhibit  the  same  granulation  and  lysis  of  the  organisms.  The 
reaction  does  not  occur  when  the  vibrios  alone  are  introduced  into 
the  peritoneal  cavity  of  a  normal  pig.  It  is  much  simpler  to  introduce 
the  immune  serum  and  vibrios  into  test-tubes,  incubate  them  at  36° 
C.  and  examine  the  contents  of  the  tubes  for  granulated  and  partly 
dissolved  organisms  after  intervals  up  to  two  hours.  The  test  is 
carried  out  as  follows:  a  series  of  dilutions  of  fresh  immune  serum, 
1  to  50  to  1  to  500,  is  prepared  in  small  sterile  test-tubes,  0.5  c.c.  to 
each  tube.  A  suspension  of  cholera  vibrios,  one  loopful  of  an  eighteen- 
hour  agar  slant  growth  to  1  c.c.  of  sterile  salt  solution,  is  also  prepared; 
usually  10  c.c.  are  sufficient.  This  is  thoroughly  shaken  and  0.5  c.c. 
added  to  each  tube  of  diluted  serum.  Control  tubes  of  normal  serum 
and  bacterial  suspension  are  incubated  uuder  parallel  conditions. 
The  entire  set  of  tubes  is  incubated  at  37°  C.  and  examined  at  intervals 
up  to  four  hours.  The  control  tubes  swarm  with  vibrios.  The  immune 
serum  tubes  uj)  to  the  limits  of  potency  contain  vibrios  in  various 
stages  of  solution.  Only  true  cholera  vibrios  will  be  thus  dissolved. 
The  various  cholera-like  vibrios  are  unaffected. 

The  bacteriological  diagnosis  of  the  cholera  vibrio  is  one  of  the 
most  difficult  known  to  bacteriology.  The  large  number  of  closely 
related  forms  introduces  complications  in  the  diagnosis  which  have 
frequently  led  to  error.  In  general  it  may  be  stated  that  a  vibrio 
which  agglutinates  T  oVo"  with  a  specific  anticholera  serum  of  high 
potency,  and  exhibits  the  Pfeiffer  phenomenon  in  a  perfectly  typical 
manner  may  be  safely  diagnosed  as  positive.  Departure  from  this 
standard  should  cause  the  organism  to  be  regarded  with  suspicion, 
but  should  not  lead  to  relaxation  of  appropriate  hygienic  measures  in 
relation  to  the  case. 


510  THE  CHOLERA   GROUP 

5.  Complement  Fixation. — Besche  and  Kon,1  Neufeld  and  Haendel,2 
and  others  have  been  successful  in  diagnosing  cholera  and  identifying 
cholera  vibrios  by  means  of  the  complement-fixation  test.    This  method 
has  not  been  generally  used,  however. 

6.  Agglutination  by  Serum  of  Patient. — The  agglutination  reaction 
is  not  of  much  value  for  an  early  diagnosis  of  Asiatic  cholera.    Agglu- 
tinins  occasionally  appear  in  the  blood  serum  of  cholera  patients 
as  early  as  the  third  or  fourth  day;  usually,  however,  they  are  not 
demonstrable  until  later.     A  dilution  of  at  least  1  to  50  should  be 
obtained  with  the  patient's  serum  to  warrant  a  positive  diagnosis. 
Even  in  chronic  cases  and  in  cholera  carriers  this  reaction  is  too 
inconstant  to  serve  practical  needs. 

Dissemination. — Cholera  vibrios  are  found  in  the  fecal  discharges 
of  cholera  patients,  but  practically  never  in  the  urine,  so  far  as  is 
known.  The  disease  is  spread  through  contaminated  water  and 
sewage,  occasionally  by  uncooked  vegetables  and  by  fomites,  rarely 
by  milk.  Dissemination  by  flies  is  probably  fairly  common,  par- 
ticularly in  those  countries,  as  India,  where  the  dejecta  are  not 
properly  disposed  of.  Those  in  contact  with  the  dejecta  of  cholera 
patients,  particularly  doctors,  nurses,  and  especially  laundresses,  are 
quite  likely  to  contract  the  disease.  The  sacred  rivers  of  India,  the 
Ganges  and  the  Jumna,  are  regarded  by  many  as  the  home  of  the 
cholera  vibrio,  and  it  has  been  accepted  in  the  past  that  drinking 
the  water  of  these  rivers  by  pilgrims  who  visited  them  in  large  numbers 
yearly  has  been  responsible  to  a  large  degree  for  the  spreading  of  the 
disease,  particularly  in  India.  Hankin3  has  made  the  astonishing 
statement  that  the  waters  of  these  rivers  kill  cholera  vibrios  in  two 
to  four  hours,  it  being  surmised  that  some  soluble  acid  substance 
is  the  bactericidal  agent.  This  observation,  if  corroborated,  would 
discredit  the  spreading  of  cholera  by  pilgrims  who  bathe  in  the  sacred 
rivers. 

Cholera  Carriers. — The  observations  of  Greig,  who  found  cholera 
vibrios  in  the  gall-bladders  of  81  out  of  271  cholera  cadavers,  and  of 
Kulescha,4  who  described  pathological  changes  in  the  gall-bladder 
and  biliary  passages  caused  by  cholera  vibrios,  have  attracted  atten- 
tion to  the  importance  of  cholera  carriers  in  the  spreading  of  the 
disease.  Zeidler5  found  cholera  organisms  in  the  feces  of  a  patient 

1  Ztschr.  f.  Hyg.,  1909,  Ixii,  161. 

2  Arb.  a.  d.  kais.  Gesundamte.,  1907,  xxvi. 

3  Ann.  Inst.  Past.,  1896,  175,  511. 

«  Centralbl.  f.  Bakt.,  1909,  1,  417.          »  Med.  Klinik.,  1907,  Nos.  48  and  49. 


CHOLERA   VIBRIO  511 

ninety-three  days  after  recovery,  suggesting  that  these  carriers  might 
be  of  hygienic  concern  for  months  after  recovery.  Zlatorgoroff1  and 
others  have  made  similar  observations.  Even  healthy  individuals 
who  are  in  contact  with  cholera  patients  may  have  cholera  organisms 
in  their  feces  without  symptoms.  It  must  be  remembered  in  this 
connection,  however,  that  curved  bacilli  morphologically  like  cholera 
organisms,  but  not  giving  specific  serum  reactions,  are  not  uncommon 
in  the  feces  of  healthy  people.  Generally  speaking,  cholera  carriers 
are  somewhat  less  likely  to  occur  than  typhoid  carriers. 

Isolation  of  Cholera  from  Water. — The  simplest  method  of  isolating 
cholera  vibrios  from  water  is  to  prepare  a  sterile  stock  solution  con- 
taining 10  per  cent,  of  peptone  and  5  per  cent,  of  salt;  to  every 
100  c.c.  of  water  to  be  examined  10  c.c.  of  this  stock  solution  are 
added,  which  practically  converts  the  suspected  water  into  a  culture 
medium.  The  isolation  then  is  carried  out  by  the  Schottelius  method 
described  above.  The  initial  culture  being  the  water  itself,  succes- 
sively inoculating  Dunham's  tubes  from  the  surface  growth  obtained 
in  the  water  culture  after  it  has  been  incubated  at  37°  C.  for  forty- 
eight  hours,  and  finally  making  agglutination  tests  with  a  high  potency 
serum  for  the  final  agglutination  of  the  organisms  is  almost  invariably 
successful. 

Vibrio  of  Finkler  and  Prior  (Vibrio  Proteus). — The  organism  was 
first  isolated  and  described  by  Finkler  and  Prior.2  .  It  was  obtained 
from  the  dejecta  of  a  case  of  acute  enteritis  and  subsequently  isolated 
from  the  dejecta  of  patients  having  cholera  nostras. 

Synonyms. — Vibrio  Proteus. — Perhaps  identical  with  Miller's  vibrio 
found  in  carious  teeth  in  1884.3 

Morphology. — Very  much  like  the  cholera  vibrio  except  that  the 
organism  is  somewhat  larger,  exhibits  a  greater  degree  of  curvature, 
and  is  said  to  have  slightly  pointed  ends.  The  organism  occurs  singly 
and  in  pairs,  rarely  in  long  spirals.  Involution  forms,  however,  are 
very  common.  There  is  a  single  polar  flagellum,  and  the  organism 
is  actively  motile.  It  stains  readily  with  the  ordinary  anilin  dyes  and 
is  Gram-negative. 

Isolation  and  Culture. — The  organism  liquefies  gelatin  with  great 
rapidity,  otherwise  there  is  nothing  characteristic  about  the  growth 
in  gelatin. 

1  Centralbl.  f.  Bakt.,  1911,  Iviii,  14. 

2  Deutsch.  med.  Wchnschr.,  1884,  x,  632-657. 

3  Miller,  Mikroorganismen  d.  Mundhohle. 


512  THE  CHOLERA   GROUP 

Growth  on  Artificial  Media. — Gelatin  stab  cultures  are  rapidly  lique- 
fied. There  is  not  the  "air  bubble"  appearance  which  is  characteristic 
of  stab  cultures  of  the  cholera  organism  ordinarily.  On  agar  there 
is  a  rapidly  spreading  growth  which  becomes  thick,  moist  and  slightly 
viscid.  Broth  is  clouded  and  there  is  a  heavy  sediment  and  a  pellicle. 
Blood  serum  is  rapidly  liquefied  and  milk  is  coagulated.  Acid  is 
formed  in  dextrose. 

Products  of  Growth. — The  nitroso  indol  reaction  is  given  very  slightly, 
frequently  not  at  all.  Indol,  however,  is  produced  in  large  amounts. 
Proteolytic  ferments  dissolving  gelatin,  serum  and  casein  are  formed 
by  Vibrio  proteus.  Cultures  have  a  foul  odor.  According  to  Kupria- 
now1  levorotatory  lactic  acid  is  formed  from  dextrose. 


FIG.  73. — Vibrio  metchnikovi,  bouillon  culture.      X  1000. 

Bacteriological  Diagnosis. — Diagnosis  depends  upon  the  isolation 
of  curved  organisms  resembling  the  cholera  vibrio,  which  do  not 
react  with  a  cholera  immune  serum. 

Pathogenesis. —  Human. — According  to  Metchnikoff,  an  agar  culture 
eaten  by  man  may  result  in  a  slight  intestinal  disturbance.  This, 
however,  probably  has  no  .significance. 

Animal. — The  intraperitoneal  inoculation  of  cultures  of  Vibrio 
proteus  causes  a  fatal  peritonitis.  According  to  Metchnikoff,2  by 
feeding  cultures  to  animals  previously  treated  with  sodium  carbonate 
and  laudanum  to  reduce  the  acidity  and  intestinal  peristalsis,  irregular 
results  are  obtained.  Occasionally  a  profuse  diarrhea  results,  but  it 
is  rarely  or  never  fatal.  In  pigeons  inoculation  into  the  pectoral 

1  Arch.  f.  Hyg.,  1893,  xix,  288. 

2  Ann.  Inst.  Past.,  1893,,  570. 


CHOLERA    VIBRIO  513 

muscles  very  frequently  produces  death.  The  organism  is  of  interest 
chiefly  because  it  is  one  of  the  classical  organisms  for  study.  It  is 
rarely  confused  with  the  cholera  vibrio  and  has  no  significance  patho- 
genically. 

Vibrio  Metchnikovi. — A  spirillum  found  in  the  feces  of  fowls  suffering 
from  acute  enteritis  by  Gamaleia.1 

Morphology. — Practically  identical  with  cholera.  Staining,  culture 
reactions,  products  of  growth,  the  same  as  cholera.  It  is  non-patho- 
genic for  man.  If  it  is  ingested  by  man  it  is  harmless.  It  does  not 
agglutinate  with  the  cholera  immune  serum,  and  is  not  dissolved  by 
the  cholera  immune  serum.  According  to  Pfeiffer  and  Nocht,2  the 
intrapectoral  injection  of  this  organism  into  pigeons  kills  them  with 
symptoms  of  acute  septicemia.  There  is  extensive  edema  at  the  site 
of  inoculation.  If  it  is  fed  to  young  fowls  it  frequently  kills  them 
with  symptoms  of  enteritis. 

Vibrio  Massaua. — Pasquale  isolated  this  organism  at  Massaua 
from  a  case  of  clinically  doubtful  cholera.3  Pathogenically  it  is  quite 
similar  to  Spirillum  metchnikovi,  and  produces  septicemia  in  birds 
when  inoculated  intrapectorally .  It  does  not  react  with  cholera  immune 
serum  either  by  agglutinating  or  by  lysis. 

Vibrio  Tyrogenum  (Spirillum  Deneke). — Deneke4  isolated  this 
organism  from  an  old  cheese,  and  it  has  since  been  found  in  butter. 
Culturally  it  is  very  similar  to  the  spirillum  of  Finkler  and  Prior, 
except  that  the  cholera-red  reaction  is  usually  negative.  Intraperi- 
toneal  injection  into  guinea-pigs  and  intrapectoral  injection  into 
pigeons  cause  death.  According  to  Metchnikoff,  a  moderate  diarrhea 
may  be  induced  in  man  by  feeding  cultures  of  this  organism. 

1  Ann.  Inst.  Past.,  1888. 

2  Ztschr.  f.  Hyg.,  1889,  vii,  259. 

3  Giorn.  Med.  de  r.  Eserc.  ed.  R.  Marina,  Roma,  1891. 

4  Deutsch.  med.  Wchnschr.,  1885,  iii. 


33 


CHAPTER  XXVII. 


TREPONEMATA  AND  SPIROCHETA. 


TREPONEMATA. 

Treponema  Pallidum. 
Treponema  Refringens. 
Treponema  Recurrentis. 
Treponema  Novyi. 
Treponema  Carter!. 


Treponema  Duttoni. 
Treponema  Pertenue. 
Treponema  Phagedenis. 
FUSIFORM  BACILLI  AND  SPIRILLUM  Fusi- 
FORMIS. 


TREPONEMATA. 


Treponema  Pallidum.— Synonym. — Spirocheta  pallida. 

Historical. — The  organism  which  is  now  universally  conceded  to 
be  the  infective  agent  in  syphilis  was  first  described  by  Schaudinn 
and  Hoffmann.1  It  was  named  Spirocheta  pallida  by  these  observers, 
but  it  presents  certain  peculiarities  of  structure  which  are  of  sufficient 
magnitude  to  separate  it  from  the  group  of  the  spirochetes.  It  has 
been  placed  in  a  newly  established  group,  the  Treponemata,  of  which 
it  is  the  type  organism. 

Morphology. — Treponema  pallidum  is  a  long,  very  thin,  delicate, 
closely  coiled,  flexous  spiral  organism  which  measures  from  0.25  to 
0.4  micron  in  diameter,  and,  on  the  average  7  to  8  microns  in 
length.  The  length,  however,  may  vary  from  3  microns  in  very  young 
organisms  to  15  microns.  The  spirals,  which  are  very  regular  in  out- 
line, are  ordinarily  from  six  to  twelve  in  number  per  organism;  they 
may  be  as  few  as  three  to  five  in  the  shorter  forms  or  as  numerous 
as  twenty  in  the  longer  forms. 

Noguchi2  has  described  three  morphologically  recognizable  types  of 
Treponema  pallidum:  an  average  or  normal  type;  a  type  thicker 
than  the  average;  and  a  type  thinner  than  the  average;  each  of 
which  induces  somewhat  different  lesions  in  experimental  animals. 
The  three  types  present  no  noteworthy  cultural  differences.  Noguchi 
suggests  that  these  morphological  and  pathological  variations  observed 

1  Arb.  a.  d.  kais.  Gesamte,  1905,  xxii;    Deutsch.  med.  Wchnschr.,  1905,  Nos.  42-43. 

2  Jour.  Exp.  Med.,  1912,  xv,  201. 


PLATE  IV 


Direct  Cultivation  of  Treponema  Pallidum.     (Noguehi.) 

FIG.  1. — Treponemata  pallida  from  a  young  pure  culture  in  ascitic  agar  tissue  medium,  four  days 
old,  at  37°  C.  Dark  field.  X  1400. 

FIGS.  2  and  3. — The  same  after  two  weeks. 

FIG.  4. — Treponemata  pallida  from  a  chancre  (for  comparison).      X  1400. 

FIG.  5. — Treponemata  pallida  from  a  pure  culture  in  ascitic  agar  tissue  medium,  two  weeks  at 
37°  C.  The  pallida  in  free  space  show  typical  morphology. 

FIG.  6. — A  short  pallidum  with  flagella-like  projections  at  both  ends.      X  1400. 

FIGS.  7,  8,  9,  and  10. — An  ascitic  agar  tissue  culture  (pure)  showing  various  phases  of  longitudinal 

division          V  1  4OO 


TREPONEMATA  515 

in  cultures  of  Treponema  pallidum  may  constitute  racial  difference 
within  the  species.1 

The  ends  of  the  organisms  are  attenuated  and  merge  almost  per- 
ceptibly into  polar  flagella,  one  at  each  end.  The  morphology  of  the 
Treponemata  varies  somewhat  in  artificial  media,  according  to  the 
conditions  of  growth.  According  to  Noguchi,  the  typical  organisms 
are  only  observed  in  special  media  where  the  conditions  of  culture 
are  strictly  anaerobic.  The  admission  of  even  slight  amounts  of 
oxygen  produces  changes  in  their  appearance.  Reproduction,  accord- 
ing to  Schaudinn2  and  Noguchi,3  takes  place  typically  by  longitudinal 
fission  rather  than  by  transverse  fission,  as  was  claimed  by  Levaditi 
and  others.  This  would  suggest  a  relationship  with  the  protozoa 
rather  than  with  the  true  bacteria. 

Treponemata  are  actively  motile  in  young  cultures,  particularly 
in  media  which  are  fluid  or  semi-fluid.  In  agar  of  ordinary  density 
the  motility  is  considerably  lessened  or  even  absent.  The  motility 
is  brought  about  by  the  activity  of  the  polar  flagella  mentioned  above. 
The  character  of  the  motion  is  twofold:  a  rotation  about  the  long 
axis,  and  a  true  progressive  motion.  The  resultant  motion  is  like 
that  of  a  corkscrew.  Undulatory  contractions  of  the  organisms  have 
also  been  observed.  No  capsules  have  been  discovered  and  no  spores 
are  produced.  It  has  been  claimed  that  an  undulatory  membrane 
has  been  demonstrated  on  Treponema  pallidum,  but  this  observation 
has  not  been  adequately  confirmed.  Treponema  pallidum  does  not 
stain  with  ordinary  anilin  dyes,  it  is  non-acid-fast  and  can  not  be 
stained  by  Gram's  method.  The  organisms  may  be  demonstrated 
in  the  living  state  on  suitable  material  scraped  from  syphilitic  lesions 
or  stained  by  special  methods  (vide  infra) . 

Isolation  and  Culture. — Various  successful  attempts  to  induce  multi- 
plication of  Treponemata,  both  in  vivo  and  in  vitro,  are  on  record. 
Brucker  and  Gelasesco4  and  Sowade5  injected  material  from  syphilitic 
lesions  into  the  testicles  of  rabbits  and  observed  considerable  prolifera- 

1  Nichols  (Jour.  Exp.  Med.,  1914,  xvii,  362)  has  described  a  Treponema  isolated  from 
the  spinal  fluid  of  a  syphilitic  which  conforms  morphologically  to  the  "thick  type"  of 
Noguchi.  It  produces  a  rapidly  developing  lesion  in  the  male  rabbit  when  inoculated 
into  the  testicle.  The  incubation  period  is  about  two  weeks  and  one-half,  and  the 
organism  tends  to  cause  generalized  secondary  lesions  in  the  eye,  and  the  skin.  It  is 
not  known  whether  this  tendency  toward  generalized  infection  is  peculiar  to  this  par- 
ticular strain  or  whether  the  thicker  organisms  possess  in  common  this  property. 

2Arb.  a.  d.  kais.  Gesamte,  1907,  xxvi,  11. 

3  Journ.  Exp.  Med.,  1912,  xv,  90. 

4  Compt.  rend.  Soc.  de  biol.,  Paris,  1910,  Ixviii,  648. 
8Deutsch.  med.  Wchnschr.,  1911,  xxxvii,  682. 


516  TREPONEMATA  AND  SPIROCHETA 

tion  of  the  organisms  there.  Schereschewsky1  grew  the  organisms 
in  impure  culture  in  anaerobic  cultures  of  gelatinized  horse  serum, 
that  is,  horse  serum  which  has  been  heated  to  60°  C.  for  some  hours. 
To  Noguchi,  however,2  belongs  the  credit  of  obtaining  Treponema 
pallidum  in  pure  culture,  and  of  demonstrating  its  etiological  rela- 
tionship to  the  disease  syphilis. 

The  medium  which  gave  the  best  results  is  prepared  in  the  following 
manner:  Two  per  cent,  slightly  alkaline  agar  is  melted  and  quickly 
cooled  to  45°  to  50°  C.  and  sterile  ascitic  or  hydrocele  fluid  is  added 
in  the  proportion  of  two  parts  of  agar  to  one  part  of  fluid.  At  the  same 
time  a  small  piece  of  sterile  tissue  from  a  rabbit's  testis  or  kidney  is 
introduced.  The  medium  is  rapidly  cooled  to  room  temperature  and 


FIG.  74. — Treponema  palliduntf. 

covered  with  a  layer  of  sterile  paraffin  oil,  2  to  3  c.c.  deep,  to  keep 
out  the  air.  The  medium  is  incubated  for  two  days  to  ensure  sterility 
and  is  then  inoculated  with  appropriate  material,  after  first  being 
certain  that  the  material  contained  the  organisms.  The  syphilitic 
tissue,  prior  to  inoculation,  is  macerated  under  sterile  conditions 
with  1  per  cent,  sodium  citrate  solution  and  then  introduced  deeply 
into  the  agar-ascitic  fluid-tissue  media.  Incubation  is  maintained  at 
37°  C.  for  two  to  three  weeks.  The  Treponemata  in  virtue  of  their 
motility  move  away  from  the  line  of  inoculation  and  cause  a  more 
or  less  uniform,  faint  clouding  of  the  medium.  The  associated  con- 
taminating organisms  are  for  the  most  part  confined  chiefly  to  the 
line  of  inoculation.  At  the  end  of  the  period  of  .incubation  the  tube 

\ 

1  Deutsch.  med.  Wchnschr.,  1909,  xxxv,  835,  1260. 

2  Jour.  Am.  Med,  Assn.,  1911,  xvii,  1.02;   Jour.  Exp.  Med.,  1912,  xv,  90. 


TREPONEMATA  517 

is  broken  at  an  appropriate  level  with  sterile  precautions  and  some 
of  the  turbid  medium  removed  into  fresh  tubes  of  the  same  kind, 
and  the  process  repeated  until  pure  cultures  of  the  organisms  are 
obtained.  If  gas-producing  bacteria  are  present  the  results  are  unsuc- 
cessful as  a  rule. 

Products  of  Growth. — The  products  of  growth  are  unknown.  Tre- 
ponema  pallidum  does  not  produce  a  characteristic  and  disagreeable 
odor  which  distinguishes  it  from  cultures  of  other  spirochetes  in 
artificial  media.1 

Pathogenesis. — Animal.  —  In  1903  Metchnikoff  and  Roux2  trans- 
mitted syphilis  to  a  chimpanzee,  and  later  infected  other  monkeys 


FIG.  75. — Treponema  pallidum,  congenital  syphilitic  liver. 

with  material  from  primary  or  secondary  lesions  in  man.  These 
results  have  been  amply  confirmed  by  other  investigators.  The 
incubation  period  averages  about  three  to  four  weeks.  It  may  be  as 
brief  as  two  weeks  or  as  prolonged  as  seven  weeks.  The  lesion,  his- 
tologically  indistinguishable  from  a  chancre,  appears  soon  after  the 
end  of  the  incubation  period  at  the  site  of  inoculation;  the  regional 
glands  become  enlarged  and  indurated.  Secondary  lesions  appear 
in  about  50  per  cent,  of  successful  inoculations,  usually  four  to  five 
weeks  after  a  chancre  appears.  Skin  lesions  are  somewhat  indefinite, 
but  the  mucous  patches  are  readily  recognized.  No  tertiary  lesions 
have  been  demonstrated  in  experimental  inoculations  into  animals 
with  the  virus  of  syphilis  up  to  the  present  time.  Recently  Noguchi3 

1  Noguchi,  Jour.  Exp.  Med.,  1912,  xv,  99. 

2  Deutsch.  med.  Wchnschr.,  1903,  No.  50. 

3  Loc.  cit.,  p.  96. 


518  TREPONEMATA  AND  SPIROCHETA 

has  successfully  inoculated  two  monkeys  (Macacus  rhesus  and  Serco- 
pithecus  callitrichus)  with  pure  cultures  of  Treponema  of  human 
origin,  and  reproduced  in  them  the  initial  lesions  of  the  disease.  The 
blood  of  these  monkeys  gave  a  positive  Wassermann  reaction,  thus 
confirming  the  relation  of  the  Treponema  pallidum  to  the  disease  in 
man.  Rabbits  have  been  successfully  infected  with  the  virus.  Bar- 
tarelli1  produced  localized  eye  lesions  by  introducing  virus  from  man 
into  the  anterior  chamber  of  the  eye.  A  small  swelling  of  the  cornea 
took  place  about  ten  days  after  inoculation  and  there  was  a  con- 
siderable development  of  Treponemata.  Brucker  and  Gelasesco2 
and  Sowade3  have  corroborated  these  results.  Hoffmann  has  pro- 
duced a  specific  orchitis  in  rabbits.  Localized  limited  growths  have 
been  reported  in  various  other  experimental  animals,  guinea-pigs, 
dogs,  sheep  and  cats.  These  inoculations  have  usually  been  made 
on  the  cornea  by  scarification,  and  slight  nodules  have  developed. 

Human. — Treponema  pallidum  is  present  in  the  hard  chancre,  in 
which  it  can  be  found  in  practically  every  case;  also  it  is  found  in 
the  enlarged  regional  glands.  The  organisms  have  also  been  found 
in  the  secondary  lesions,  particularly  in  the  mucous  patches  and 
papules.  According  to  Bandi  and  Simmonelli,4  the  organisms  are 
occasionally  found  in  the  blood,  and  they  have  also  been  observed  in 
blister  fluid  by  Levaditi  and  Petresco.5 

In  the  lesions  of  tertiary  syphilis  the  organisms  are  present  in  but 
small  numbers,  although  usually  these  lesions  are  infective  for  mon- 
keys. Noguchi  has  found  the  organisms  in  the  cerebral  cortex  in 
many  cases  of  general  paresis,  and  Reuter  has  demonstrated  the 
organisms  in  the  walls  of  the  larger  bloodvessels  in  an  individual 
infected  with  syphilitic  aortitis.  The  organisms  are  present  in 
enormous  numbers  in  the  liver,  spleen  and  internal  organs  of  cases 
of  congenital  syphilis. 

Bacteriological  Diagnosis. — Collection  of  Material — The  distribution 
of  the  organism  in  syphilitic  tissues  is  quite  irregular,  the  organisms 
being  very  numerous  in  some  cases,  in  other  apparently  similar  cases 
so  few  in  number  that  they  may  be  readily  overlooked.  In  congenital 
syphilis  the  organisms  are  extremely  numerous;  in  the  lesions  of 
acquired  syphilis  the  organisms  are  best  observed  either  in  the  primary 
or  secondary  stages. 

1  Centralbl.  f.  Bakt.,  1906,  xli,  320.  2  Loc.  cit. 

3  Deutsch.  med.  Wchnschr.,  1911,  xxxvii,  1540. 

4  Centralbl.  f.  Bakt.,  1905,  xl,  64. 

5  Presse  M6dicale,  1905. 


TREPONEMATA  519 

Primary  Lesion. — Clean  the  surface  of  the  chancre  with  brisk  rub- 
bing, then  make  an  abrasion  in  the  skin  deep  enough  so  that  there  is 
an  exudation  of  serum.  Films  are  prepared  from  this  exudate. 

Secondary  Lesion;  Mucous  Patches  and  Papules. — Material  is 
removed  from  the  mucous  patch  after  cleaning  the  surface,  or  from 
the  papule  by  slight  curetting.  If  the  material  thus  obtained  is  too 
dense  to  spread  readily  it  may  be  macerated  in  a  drop  or  two  of 
sterile  ascitic  fluid. 

1.  Morphology. — The  organisms  may  be  seen  in  the  living  state 
with  the  dark-ground  illuminating  apparatus.    The  juice  of  a  mucous 
patch  or  primary  lesion  is  examined  directly  and  the  organisms  appear 
on  a  black  background  as  light  yellowish  closely  coiled  spirals,  which 
are  actively  motile.    The  presence  of  Spirocheta  (Treponema)  refrin- 
gens  must  be  borne  in  mind,  this  organism  being  frequently  associated 
with  Treponema  pallidum.     The  former  is  thicker  than  Treponema 
pallidum,  and  the  spirals  are  less  numerous  and  coarser. 

India  Ink  Method. — The  juice  from  a  chancre  or  mucous  patch  is 
intimately  mixed  with  india  ink1  and  a  cover  glass  placed  over  the 
mixture.  The  organisms  appear  as  white  spirals  against  a  black 
background. 

Other  Staining  Methods. — The  material  collected  as  above  is  spread 
on  slides  in  thin  layers  and  stained  either  by  Schaudinn  and  Hoffmann's 
original  method  or  by  the  silver  impregnation  method. 

Method  of  Schaudinn  and  Hoffmann. — The  films  are  fixed  for  fifteen 
to  thirty  minutes  in  absolute  methyl  alcohol  or  for  a  few  seconds 
in  the  vapor  of  osmic  acid,  then  they  are  stained  from  one  to  three 
hours  in  the  following  solution,  which  must  be  freshly  prepared  each 
time;  Giemsa's  solution,  10  drops;  1  per  cent,  aqueous  solution  of 
potassium  carbonate,  10  drops;  distilled  water,  10  c.c.  The  films, 
after  staining,  are  washed  in  distilled  water.  If  overstaining  has 
taken  place  the  film  may  be  left  in  distilled  water  for  some  minutes 
until  a  sufficient  amount  of  stain  has  been  removed.  The  preparation 
is  then  dried  and  examined.  The  organisms  appear  as  purple  or  violet 
spirals  on  a  bluish  background. 

Silver  Impregnation  Method.2    Not  generally  used. 

2.  Cultural  Diagnosis.    Not  practical  for  routine. 

3.  Serum  Diagnosis.    (For  Technic  see  page  161.) 


1  Burn,  Wien.  klin.  Wchnschr.,  July  1,  1909. 

2  See  Levaditi,  Compt.  rend,  de  Soc.  de  Biol.,  1905,  lix,  326,  for  details. 


520  TREPONEMATA   AND  SPI  ROCHET  A 

For  a  time  the  specificity  of  the  Wassermann  reaction  for  syphilis 
was  questioned,  because  it  was  found  that  alcoholic  extracts  of  normal 
heart  could  be  substituted  for  extracts  of  luetic  organs  as  antigens. 
A  careful  study  of  thousands  of  cases  has  shown,  however,  that  a  vast 
majority  of  active  syphilitic  infections,  especially  those  in  the  second- 
ary stage,  give  a  positive  Wassermann  reaction.  During  the  earlier 
part  of  the  primary  stage  the  reaction  is  frequently  negative.  In 
the  tertiary  stage  the  reaction  is  frequently  positive  and  the  spinal 
fluid  frequently  gives  a  positive  reaction  as  well.  Occasionally  cases 
of  frambesia  and  of  leprosy  give  a  positive  Wassermann  reaction, 
but  these  diseases  are  rare  in  temperate  climates.  For  a  time  it  was' 
believed  that  the  serum  in  cases  of  scarlet  fever  gave  a  Wassermann 
reaction,  but  this  view  has  not  been  fully  substantiated. 

Statistics  indicate  that  the  Wassermann  reaction  disappears  when 
a  cure  is  effected,  but  it  reappears  if  the  disease  again  becomes  active. 
It  is  important  to  remember  that  the  mercurial  treatment  tends  to 
diminish  the  intensity  of  the  reaction,  and  it  may  even  disappear 
temporarily.  Treatment  with  salvarsan  and  neosalvarsan  may  accen- 
tuate the  reaction,  temporarily  at  least. 

There  is  no  doubt  that  the  Wassermann  reaction  carefully  executed 
by  competent  workers  is  the  most  delicate  and  reliable  diagnostic 
method  for  syphilis  known  at  the  present  time. 

4.  Luetin  reaction.1 

Preparation  of  Luetin. — A  culture  of  Treponema  pallidum  is  ground 
until  the  organisms  are  thoroughly  disrupted,  then  heated  to  60°  C. 
for  an  hour  and  suspended  in  sterile  salt  solution  to  which  is  added 
0.5  per  cent,  carbolic  acid  as  a  preservative.  The  reaction  induced 
in  syphilitics  is  essentially  like  the  tuberculin  reaction.  It  consists 
in  the  development  of  a  vesicle  or  a  pustule  at  the  site  of  inoculation 
with  temperature  and  pain.  The  control  inoculation  should  exhibit 
but  a  slight  reddening.  The  reaction  is  specific,  except  for  old, 
advanced  cases,  where  the  reaction  may  fail.  It  is  most  marked  in 
the  later  tertiary  and  congenital  cases  where  the  Wassermann  reac- 
tion is  said  to  be  more  likely  to  be  negative. 

Clinical  Methods  of  Serum  Diagnosis. — Method  of  Forges  and  Meyer.— 
The  principle  of  this  reaction  depends  on  the  production  of  a  precipi- 
tate when  syphilitic  serum  is  mixed  with  lecithin.  Normal  serum  does 
not  produce  a  precipitate  under  these  conditions.  The  technic  con- 
sists in  thoroughly  triturating  0.25  grams  of  lecithin  (ovolecithin)  in 

1  Noguchi,  Jour.  Exp.  Med.,  1911,  xiii,  557. 


PLATE  V 


Cultivation  of  Spiroeheta  Refringens.     (Noguehi.) 

FIG.  1. — A  schematic  drawing  of  Spiroeheta  refringens  from  pure  cultures  (dark  field). 
FIG.  2. — Spirocheta  refringens  from  a  three  weeks'  old  pure  culture  in  ascitic  tissue  agar.  at 
37°  C.  (dark  field).      X  1100. 

FIG.  3. — Spirocheta  refringens  from  a  pure  culture  seven  days  old  (dark  field).       X  1100. 
FIGS.  4  and  5. — Spirocheta  refringens  from  a  pure  culture  two  weeks  old  (dark  field).     X  1100. 


TREPONEMATA  521 

100  c.c.  of  normal  saline  solution.  One  c.c.  of  this  lecithin  emulsion 
is  added  to  each  of  a  number  of  test  tubes  of  5  mm.  diameter.  To  half 
of  the  tubes  add  1  c.c.  of  the  suspected  syphilitic  serum;  to  the  remain- 
ing tubes  add  to  some  a  known  syphilitic  serum,  to  others  normal 
serum,  and  incubate  the  entire  number  about  four  hours  at  37°  C. 
The  tubes  are  then  removed  from  the  incubator,  cooled  to  room 
temperature,  and  those  containing  syphilitic  serum  will  show  a  pre- 
cipitate, which  appears  to  develop  first  at  the  surface.  It  is  best 
observed  against  a  dark  background.  This  method  has  been  modified 
by  Forges  by  the  substitution  of  a  solution  of  sodium  glycocholate 
for  the  lecithin.  A  freshly  prepared,  1  per  cent,  solution  of  sodium 
glycocholate  is  made  in  distilled  water.  The  test  is  carried  out  pre- 
cisely as  in  the  above  method,  except  that  the  suspected  serum,  the 
known  serum,  and  the  normal  serum  are  heated  to  55°  C.  for  thirty 
minutes  before  being  added  to  the  solution.  One  c.c.  each  of  heated 
serum  and  sodium  glycocholate  are  mixed  together  and  kept  at  room 
temperature  for  twenty-four  hours.  A  precipitate  forms  at  the  sur- 
face of  the  tubes  containing  the  syphilitic  serum,  but  does  not  form 
in  the  tubes  containing  normal  serum. 

Treponema  Refringens. — Synonym. — Spirocheta  refringens. 

Schaudinn  and  Hoffmann1  observed  Treponema  refringens  both  in 
syphilitic  lesions  in  association  with  Treponema  pallidum,  and  in 
non-syphilitic  lesions  as  well,  particularly  in  superficial  lesions  of  the 
genitalia.  This  association  of  Treponema  refringens  with  Treponema 
pallidum  in  syphilitic  lesions  and  its  common  occurrence  in  non- 
specific genital  lesions  emphasize  the  necessity  of  its  recognition  and 
differentiation  from  Treponema  pallidum. 

Morphology. — Observed  under  the  dark-field  microscope,  Treponema 
refringens  is  noticeably  thicker  than  Treponema  pallidum,  measuring, 
according  to  Noguchi,2  0.5  to  0.75  micron  in  diameter  and  6  to  24 
microns  in  length.  The  ends  are  somewhat  sharply  attenuated  and 
they  are  continued  as  moderately  stiff,  delicate  spiral  flagella.  Not 
infrequently  the  middle  third  of  the  organism  is  slightly  wavy  in 
outline,  the  end  thirds  being  more  closely  coiled.  Usually  the  spiro- 
chetes  are  more  uniformly  curved.  Occasionally  two  or  three  organisms 
may  be  joined  end  to  end.  As  a  rule  there  are  from  three  to  eight 
complete  spirals  in  each  organism. 

The  organisms  are  actively  motile,  and  observed  with  the  dark- 

1  Arb.  a.  d.  kais.  Gesamte,  1905,  xxii,  Heft  2. 

2  Jour.  Exp.  Med.,  1912,  xv,  467. 


522  TREPONEMATA  AND  SPIROCHETA 

field  illumination  they  are  golden  yellow,  contrasting  in  this  respect 
with  the  pale  yellow  appearance  of  Treponema  pallidum.  The  stain- 
ing reactions  are  similar  to  those  of  Treponema  pallidum. 

Isolation  and  Culture. — Noguchi  was  the  first  to  grow  Treponema 
refringens  in  pure  culture  using  the  ag'ar  ascitic  fluid  tissue  medium 
with  which  he  isolated  Treponema  pallidum.  The  organism  is  a 
strict  anaerobe,  but  it  may  be  obtained  in  the  agar  ascitic  medium 
without  the  sterile  tissue,  although  the  growth  is  much  more  feeble 
when  the  tissue  is  omitted  from  the  medium.  The  original  growths 
from  lesion  to  artificial  media  are  usually  contaminated  with  other 
organisms.  Purification  is  accomplished  by  the  same  technic  as  that 
used  for  purifying  Treponema  pallidum.  Pure  cultures  of  Treponema 
refringens  produce  no  odor  in  growths  on  artificial  media. 


FIG.  76. — Treponema  recurrentis.     (Kolle  and  Hetsch.) 

Pathogenesis. — The  organism  was  found  to  be  non-pathogenic  for 
rabbits  and  monkeys.1 

Relapsing  Fever. — The  disease  known  as  relapsing  fever  was 
described  by  Obermeier  in  1878;  he  recognized  the  organism  which 
received  his  name,  Spirocheta  obermeieri,  now  called  Treponema 
recurrentis,  in  the  blood  of  his  patients.  Obermeier's  observations 
were  made  in  Europe.  Somewhat  later  the  disease  was  observed  in 
India  by  Carter,  in  Africa  by  Koch,  and  in  America  by  Norris,  Pap- 
penheimer  and  Fluornoy.  In  1896  Novy  showed  that  the  organisms 
found  respectively  in  the  relapsing  fevers  of  Europe,  India,  Africa 
and  America  exhibited  constant  morphological  differences  which  war- 
rant their  tentative  separation  into  four  distinct  types :  the  European, 

Uour.  Exp.  Med.,  1912,  xv,  90. 


TREPONEMATA  523 

Indian,  African  and  American.  Noguchi  grew  the  organisms  in  pure 
culture  for  the  first  time  in  1912.  Relapsing  fever  appears  to  be 
transmitted  chiefly,  if  not  exclusively,  by  suctorial  insects. 

Treponema  Recurrentis. — Synonyms. — Spirillum  obermeieri,  Spiro- 
cheta  obermeieri,  Spirillum  recurrentis,  Treponema  obermeieri,  Spiro- 
cheta  recurrentis. 

Relapsing  fever  is  an  acute  contagious  disease  which  begins  abruptly 
with  a  chill.  The  fever  which  follows  immediately  after  the  chill 
reaches  the  fastigium  (104°  to  106°)  usually  within  twenty-four  hours, 
remains  high  for  five  to  seven  days,  and  falls  by  crisis.  There  is  an 
afebrile  intermission  of  five  to  seven  days,  then  the  fever  is  repeated.1 
Convalescence  usually  begins  at  the  close  of  the  second  paroxysm; 
it  may  not  occur  until  the  close  of  the  third  or  even  fourth  paroxysm. 
The  incubation  period  is  from  two  to  fourteen  days.  The  mortality 
is  low,  less  than  4  per  cent,  of  all  cases.  The  spleen  is  enlarged,  there 
is  profuse  sweating,  frequently  jaundice,  and  occasionally  diarrhea. 

Morphology. — The  organism  is  spiral  in  outline  and  of  moderate 
size.  Schellack2  states  that  the  average  diameter  is  0.4  micron;  the 
length  varies  from  15  to  20  microns.  Other  investigators  give  as 
measurements,  diameter  0.25  micron,  length  from  7  to  10  microns. 
The  discrepancy  appears  to  be  attributable  to  the  fact  that  younger 
organisms  are  about  10  microns  in  length,  the  older  forms  being  much 
longer.  There  are  from  twenty  to  forty  spirals  in  each  individual 
cell,  the  number  depending  upon  its  length.  Very  frequently  the 
ends  are  tapered.  Fresh  preparations  viewed  by  dark-field  illumination 
exhibit  three  distinct  types  of  motion:  3,  rotation  around  the  long 
axis,  which  causes  the  organism  to  move  rapidly  through  the  medium 
in  which  it  is  suspended,  an  undulatory  movement,  and  a  lateral 
movement  in  all  planes.  The  motility  is  caused  by  the  rhythmic 
contractions  of  a  terminal  flagellum,  according  to  Novy  and  Knapp.3 
Zettnow4  believes  the  organism  possesses  peritrichic  flagella.  This 
has  not  been  confirmed.  Reproduction  takes  place  typically  by  longi- 
tudinal fission  according  to  Noguchi.5  Less  commonly  he  has  observed 
transverse  fission. 

Isolation  and  Culture. — The  organism  appears  in  the  blood  stream 
only  during  the  pyrexia.  Novy  and  Knapp6  observed  multiplication 

1  Obermeier,  Centralbl.  f.  d.  med.  Wissensch.,  1873,  xi;    Berl.  klin.  Wchnschr.,  1873, 
x,   152,  378,  391,  455. 

2  Arb.  a.  d.  kais.  Gesamte,  1908,  xxvii,  364.      3  Jour.  Inf.  Dis.,  1906,  iii,  291. 

4  Deutsch.  med.  Wchnschr.,  1906,  xxxi.  «  jour.  Exper.  Med.,  1912,  xvi,  207. 

6  Jour.  Am.  Med.  Assn.,  1906,  xlvii,  2152 


524  TREPONEMATA  AND  SPI  ROCHET  A 

of  Treponema  recurrentis  in  defibrinated  rat's  blood  and  succeeded 
in  keeping  these  organisms  alive  on  blood  agar  for  forty  days,  at  the 
end  of  which  time  they  were  still  infective  for  rats.  No  actual  mul- 
tiplication, however,  was  observed  in  this  medium.  Noguchi1  has 
grown  the  organisms  in  pure  culture,  using  the  method  described 
previously  (see  Treponema  pallidum).  The  organisms  develop  with 
considerable  rapidity,  a  distinct  clouding  of  the  medium  being 
observed  after  twenty-four  to  forty-eight  hours'  incubation  at  37°  C. 
The  maximum  growth  is  reached  at  the  end  of  a  week. 

Pathogenesis. — Animal. — Pure  cultures  retain  their  original  virulence 
for  rats  and  mice  for  several  transfers  in  the  agar  ascitic  fluid  tissue 
medium  described  by  Noguchi.  The  lesions  produced  in  experimental 
animals  are  essentially  the  same  as  those  observed  in  man.  The  disease 
can  be  transmitted  by  inoculation  from  man  to  monkeys,  from  monkey 
to  monkey,  and  from  monkey  to  mice  and  rats,  which  are  all  suscep- 
tible. Rabbits  and  guinea-pigs  appear  to  be  refractory.  The  disease 
produced  by  inoculation  of  the  organisms  in  monkeys  and  mice 
exhibits  the  characteristic  relapses,  and  it  may  be  fatal. 

Human. — There  are  no  characteristic  lesions  observed  in  relapsing 
fever  other  than  a  hyperplastic  enlargement  of  the  spleen.  There 
may  be  a  catarrhal  inflammation  of  the  stomach,  bile  ducts  and  liver, 
which  is  usually  enlarged.  All  of  the  organs  exhibit  parenchymatous 
degeneration  postmortem. 

Bacteriological  Diagnosis. — The  organisms  are  found  in  the  blood 
stream  only  during  the  paroxysms.  During  the  period  of  apyrexia 
they  disappear  from  the  blood  stream,  but  are  found  in  the  spleen 
in  large  numbers,  where  they  are  engulfed  by  leukocytes. 

Immunity. — According  to  Novy,2  blood  drawn  from  a  patient  at 
the  beginning  of  the  fever  acts  as  a  good  culture  medium  for  the 
organisms;  that  drawn  at  the  end  of  a  paroxysm  or  after  recovery  from 
the  disease  appears  to  possess  germicidal  properties  for  the  organisms. 
It  is  supposed  that  the  organisms  are  taken  up  by  phagocytes  during 
the  afebrile  periods,  and  that  they  are  either  weakened  or  killed  at 
this  time.  Active  immunity  follows  recovery  from  the  infection.  It 
has  been  claimed  that  the  blood  serum  of  immunized  animals  (which 
exhibit  immunity  after  repeated  injections  of  the  organism)  or  of 
animals  which  have  recovered  from  an  attack  will  induce  passive 
immunity  and  temporarily  prevent  infection  when  it  is  introduced 
into  susceptible  animals  prior  to  inoculation  of  the  organisms. 

1  Loc.  cit.,  p.  208.  2  Jour.  Inf.  Dis.,  1906,  iii,  291. 


TREPONEMATA  525 

Transmission. — The  disease  appears  to  be  transmitted  by  suctorial 
insects.  Mackie1  believes  the  human  louse,  Pediculus  vestimenti,  is 
commonly  the  one  involved,  but  Manteufel2  has  produced  evidence 
suggesting  the  rat  louse,  Hematopinus  spinosus,  is  at  times  a  carrier 
of  the  organism. 

Treponema  Novyi. — Norris,  Pappenheimer  and  Fluornoy3  appear 
to  have  been  the  first  to  report  relapsing  fever  in  America.  Several 
cases  were  studied;  the  incubation  period  averaged  from  five  to  seven 
days,  and  the  mortality  varied  from  2  to  6  per  cent.  Novy  and 
Knapp4  studied  the  organisms  in  detail  and  discovered  slight  but 
constant  differences  which  distinguished  them  from  Treponema 
recurrentis  and  Treponema  duttoni.  Schellack5  named  the  organism 
Spirocheta  novyi.  Mackie6  was  able  to  differentiate  Treponema 
novyi  from  Treponema  duttoni  by  agglutination  reactions,  and 
Manteufel7  showed  that  the  serum  of  patients  infected  with  the 
organism  of  American  relapsing  fever  did  not  agglutinate  Treponema 
recurrentis  and  vice  versa,  thus  confirming  Novy  and  Knapp's  obser- 
vations. Noguchi8  grew  the  organism  in  pure  culture. 

Treponema  Carter!. — The  causative  organism  of  the  relapsing  fever 
of  India.  In  1879  Carter9  observed  the  organism  originally  named 
Spirocheta  carteri,  but  now  known  as  Treponema  carteri,  in  the 
blood  of  patients  suffering  with  Indian  relapsing  fever,  and  he  suc- 
ceeded in  inoculating  mice  with  the  organism.  Novy  and  Knapp10 
have  shown  that  this  organism  differs  from  those  of  the  European, 
African  and  American  relapsing  fevers. 

According  to  Schellack,11  Treponema  carteri  measures  from  0.3  to 
0.35  micron  in  diameter  and  from  15  to  20  microns  in  length.  The 
organism  has  not  been  grown  in  pure  culture. 

Treponema  carteri  is  infective  for  rats  and  for  experimental  animals, 
but  it  typically  causes  but  one  relapse,  contrasting  in  this  respect 
with  the  organisms  of  the  American,  European,  and  African  relapsing 
fevers  respectively.  It  also  differs  from  the  other  Treponemata  in 
its  agglutination  reactions.12 


1  Brit.  Med.  Jour.,  December  14,  1907.        2  Arb.  a.  d.  kais.  Gesamte,  xxxix,  No.  2. 
3  Jour.  Inf.  Dis.,  1906,  iii,  266.  *  Ibid.,  p.  291. 

5  Arb.  a.  d.  kais.  Gesamte,  1908,  xxvii,  364. 

6  British  Med.  Jour.,  December  14,  1907. 

7  Arb.  a.  d.  kais.  Gesamte,  1908,  xxvii,  327.  8  Jour.  Exp.  Med.,  1912,  xvi,  208. 
9  Deutsch.  med.  Wchnschr.,  1879,  v,  189,  351,  386. 

10  Jour.  Inf.  Dis.,  1906,  iii,  291. 

11  Arb.  a.  d.  kais.  Gesamte,  1908,  xxvii,  364. 

12  Manteufel,  Arb.  a.  d.  kais.  Gesamte,  1908,  xxvii,  327. 


526  TREPONEMATA   AND  SPI  ROCHET  A 

Treponema  Duttoni. — Synonym. — Spirocheta  duttoni. 

Ross  and  Milne,1  studying  South  African  tick  fever,  observed  an 
organism  in  the  blood  of  their  patients  which  they  called  Spirocheta 
duttoni.  Button  and  Todd2  confirmed  the  discovery.  The  disease 
runs  a  course  clinically  like  European  relapsing  fever,  but  the 
paroxysms  usually  number  four  or  five  with  corresponding  periods  of 
apyrexia  before  the  onset  of  convalescence. 

Morphology. — Treponema  duttoni  (Spirocheta  duttoni)  is  somewhat 
thicker  and  longer  than  Treponema  recurrentis;  it  measures  about 
0.45  to  0.50  micron  in  diameter  and  from  24  to  30  microns  in  length. 
The  motility  is  similar  to  that  of  the  organism  of  European  relapsing 
fever.  Noguchi3  has  grown  Treponema  duttoni  in  pure  culture. 

Immunity. — Rats  are  readily  infected  with  the  organism;  those 
which  have  recovered  from  infection  with  Treponema  duttoni  are 
easily  infected  with  Treponema  recurrentis  and  vice  versa.  They 
are  refractory  to  a  second  injection  of  the  same  organism,  indicating 
that  the  immunity  conferred  by  one  Treponema  is  not  protective 
against  infection  with  Treponemata  of  another  type. 

Ross4  found  that  the  horse  tick  (Ornithodorus  moubata)  would 
transmit  the  disease  from  man  to  monkey,  provided  the  insect  bit  the 
man  during,  or  very  shortly  before,  the  febrile  period.  The  organism 
may  be  demonstrated  in  the  ovaries  and  eggs  of  female  ticks  which 
have  fed  upon  man.  This  appears  to  be  a  case  of  true  hereditary 
transmission;  the  organism  is  transmissible  by  the  adult  and  larval 
insects,  and  through  the  eggs  as  well. 

Treponema  Pertenue. — Synonyms. — Spirillum  pertenue.  Treponema 
pallidulum. 

Castellan i5  has  reported  the  constant  association  of  an  organism 
which  he  called  Spirillum  pertenue,  in  frambesia  tropica  (Yaws). 
Frambesia  is  a  specific  infectious  tropical  disease  characterized  anato- 
mically by  peculiar  specific  granulomatous  eruptions.  The  disease, 
like  syphilis,  presents  three  stages:  (1)  a  primary  lesion,  which  is  a 
papule  situated  at  the  site  of  infection — this  papule  becomes  indurated 
and  may  ulcerate;  (2)  a  generalized  eruption,  papular  in  character, 
which  gives  rise  to  characteristic  granulomata;  this  may  appear 
after  the  primary  lesion  has  healed — the  disease  frequently  ends  at 

1  British  Med.  Jour.,  1904,  ii,  1453. 

2  Ibid.,  1905,  ii,  1295. 

3  Jour.  Exp.  Med.,  1912,  xvi,  202. 

*  British  Med.  Jour.,  February  4,  1905. 

5  Lancet,  August,  1905;    British  Med.  Jour.,  November,  1905, 


TREPONEMATA  527 

the  second  stage;    (3)   tertiary  stage,  characterized  by  gumma-like 
processes  which  may  undergo  deep  ulceration. 

Morphology. — Treponema  pertenue  is  a  very  delicate,  slender 
spiral  organism,  measuring  about  0.30  to  0.50  micron  in  diameter, 
and  from  6  to  18  microns  in  length.  The  ends  of  the  organism  are 
frequently  pointed,  but  one  or  both  ends  may  be  rounded,  or,  rarely, 
somewhat  swollen.  There  are  usually  from  six  to  twenty  spiral  turns 
in  each  organism.  Blanchard1  states  that  the  organism  possesses  an 
undulatory  membrane,  but  the  consensus  of  opinion  is  against  this 
view.  Very  delicate  polar  flagella,  one  at  each  end,  have  been  demon- 
strated by  flagella  stains.  It  will  be  observed  that  the  size  and  arrange- 
ment of  the  organism  do  not  differ  essentially  from  that  of  Treponema 
pallidum.  The  organism  fails  to  stain  by  ordinary  methods,  but  the 
morphology  is  well  brought  out  by  Giemsa's  stain.  Treponema  per- 
tenue may  be  demonstrated  by  the  methods  applicable  for  Treponema 
pallidum.  It  has  never  been  cultivated  in  artificial  media. 

Specificity  of  Organism. — Paulet2  inoculated  fourteen  negroes  with 
the  secretion  from  granulomata  and  all  developed  yaws,  the  initial 
lesion  appearing  at  the  site  of  inoculation.  There  is  a  possibility  that 
these  negroes  might  have  been  naturally  infected,  however.  Charlouis 
injected  thirty-two  Chinese  prisoners  with  scrapings  from  the  granulo- 
mata of  a  case  of  yaws  and  twenty-eight  developed  the  disease,  the 
primary  lesion  again  appearing  at  the  site  of  inoculation.  This  series 
is  suggestive,  but  not  conclusive,  because  the  possibility  of  natural 
infection  can  not  be  ruled  out. 

According  to  Castellani,3  yaws  and  syphilis  are  distinct  diseases, 
because  a  native  who  had  been  inoculated  successfully  with  yaws  was 
subsequently  infected  with  material  from  a  chancre;  this  resulted  in 
a  typical  attack  of  syphilis  superimposed  upon  the  yaws.  In  Ceylon 
syphilis  is  not  uncommonly  observed  in  cases  of  yaws  which  are  in 
the  secondary  or  tertiary  stages. 

Pathogenesis. — Animal. — The  disease  may  be  transferred  to  monkeys 
by  direct  inoculation.  The  organisms  are  found  in  the  lesions. 

Human. — The  distribution  of  Treponema  pertenue  in  the  lesions 
of  yaws  is  somewhat  different  from  that  of  Treponema  pallidum  in 
syphilis.  In  the  former  the  organisms  are  numerous  in  the  spaces 
between  the  papillary  pegs  of  the  malpighian  layer  of  the  epidermis, 

1  Arch.  d.  Parasit.,  1906. 

2  Quoted  by  Castellani  and  Chalmers,  Manual  of  Tropical  Medicine. 

3  Loc.  cit. 


528 


TREPONEMATA   AND  SPI  ROCHET  A 


not  necessarily  in  intimate  association  with  bloodvessels;  in  syphilis 
the  organisms  are  found  in  considerable  numbers  around  thickened 
arteries.  Treponema  pertenue  is  found  constantly  in  the  primary 
lesion  and  in  unbroken  papules  of  the  generalized  eruption  charac- 
teristic of  the  secondary  stage  of  yaws.  In  broken  down  lesions  many 
bacteria,  including  Treponemata  indistinguishable  from  Treponema 
refringens,  complicate  the  picture.  They  are  frequently  not  found  in 
the  tertiary  stage.  At  autopsy  the  spleen,  lymph  glands  and  bone 
marrow  contain  many  Treponemata  as  a  rule;  the  cerebrospinal 
fluid  is  free  from  them  ante-  or  postmortem. 

The  disease  is  transmissible  by  direct  contact,  and  it  is  probable 
that  the  virus  may  be  transmitted  by  biting  insects  as  well. 


FIG.  77. — Treponema  balanitidis.     (Corbus.) 

Treponema  Phagedenis. — Synonym. — Spirocheta  balanitidis. 

Schaudinn  and  Hoffmann,1  Miihlens,2  Hoffmann  and  Prowazek3 
and  others  have  described  spiral  organisms  resembling  Treponema 
refringens  in  size,  shape  and  motility  in  genital  and  perigenital  ulcer a- 
tions  and  in  phagedenic  ulcers.  Similar  organisms  have  been  observed 
in  noma.  Corbus  and  Harris4  and  Corbus5  have  described  a  spiral 
organism  resembling  Vincent's  spiral  in  several  cases  of  erosive  and 
gangrenous  balanitis,  and  Brault6  has  observed  a  similar  spiral  asso- 
ciated with  a  fusiform  bacillus  in  two  cases  of  noma.  The  identity 
of  the  various  organisms  is  as  yet  undetermined,  and  their  etiological 

1  Arb.  a.  d.  kais.  Gesamte,  1905,  xxii,  Heft  2. 

2  Centralbl.  f.  Bakt.,  Orig.,  1907,  xlii,  277. 

3  Ibid.,  1906,  xli,  741,  817. 

4  Jour.  Am.  Med.  Assn.,  1909,  lii,  1474. 

6  Ibid.,  1913,  Ix,  1769.  6  Bull.  Derm,  et  Syph.,  1908,  2. 


TREPONEMATA  529 

relationship  to  genital  ulcer ations,  phagedenic  ulcers,  and  noma  is 
not  satisfactorily  established.  Noguchi1  has  isolated  a  spiral  organism 
in  pure  culture  from  a  phagedenic  ulcer,  using  the  technic  employed 
by  him  for  cultivation  of  Treponema  pallid  urn.  This  organism  is  the 
only  member  of  the  group  observed  in  genital  ulcerations  and  phage- 
denic ulcers  which  has  been  satisfactorily  studied  up  to  the  present 
time. 

Morphology. — The  organism  measures  about  0.75  micron  in  diameter 
and  about  15  microns  in  length,  although  the  length  varies  between 
the  limits  of  4  and  30  microns.  The  number  of  spirals  varies  materially 
in  different  organisms  in  the  same  culture,  from  two  complete  turns 
to  as  many  as  eight.  The  ends  of  the  organisms  are  found  to  be 
distinctly  pointed,  but  not  attenuated.  In  young  cultures  the  organ- 
isms were  found  to  be  fairly  uniform  in  size,  from  10  to  15  microns 
long.  In  older  growths  the  length  is  greater  on  the  average,  varying 
from  20  to  30  microns.  The  number  of  spiral  turns  and  the  spiral 
turns  themselves  are  more  irregular  in  the  older  growths.  This 
organism  appears  to  be  devoid  of  a  terminal  flagellum  or  a  terminal 
projection.  In  very  old  cultures  signs  of  degeneration  appear,  and 
spherical  bodies  measuring  about  0.5  micron  in  diameter  are  found 
either  attached  to  degenerating  organisms  or  free.  These  spherical 
bodies  do  not  take  the  spore  stain.  In  addition  various  semi-spherical 
bodies,  some  exhibiting  refractile  dots  in  their  substance,  are  also 
found  in  old  cultures,  but  none  of  these  bodies  appear  to  be  spores  in 
the  ordinary  sense. 

Treponema  phagedenis  stains  with  difficulty  by  the  more  penetrat- 
ing anilin  dyes,  and  it  is  Gram-negative.  It  is  colored  red  with  the 
Giemsa  stain.  The  organism  is  obligately  anaerobic,  and  cultures  in 
artificial  media  develop  an  odor  suggesting  butyric  acid. 

Pathogenesis. — Noguchi  found  that  pure  cultures  of  Treponema 
phagedenis  produce  an  acute  inflammatory  reaction  at  the  site  of 
inoculation  (intradermal)  both  in  monkeys  and  rabbits,  but  this 
inflammatory  area  does  not  ulcerate.  Hoffmann  and  Prowazek2 
inoculated  two  monkeys  with  material  from  a  case  of  balanitis  rich  in 
organisms.  They  found  some  erosion  had  taken  place  at  the  site  of 
inoculation  after  two  to  three  days,  with  numerous  spiral  organisms 
in  the  lesion.  Noguchi  did  not  consider  that  his  observations  estab- 
lished the  relationship  of  his  organism  to  the  lesion,  and  the  experiments 
of  Hoffmann  and  Prowazek  are  not  conclusive. 

1  Jour.  Exp.  Med.,  1912,  xvi,  261.  2  Loc.  cit.,  p.  818. 

34 


530  TREPONEMATA  AND  SPIROCHETA 

FUSIFORM   BACILLI   AND    SPIRILLUM   FUSIFORMIS. 

Fusiform  bacilli,  frequently  in  association  with  spiral  organisms, 
have  been  observed  by  Plaut1  and  Vincent2  in  diphtheroid  angina; 
by  Vincent3  in  cases  of  hospital  gangrene;  by  Bernheim4  in  stomatitis 
ulcerosa  and  angina  ulcerosa;  in  noma5  and  in  erosive  and  gangrenous 
balanitis  by  Corbus.6 

The  organism,  Bacillus  fusiformis,  is  a  long,  thin  bacillus  with 
distinctly  tapering  ends  measuring  from  0.5  to  0.8  micron  in  diameter 
at  the  centre,  and  varying  in  length  from  3  to  10  microns.  The  bacilli 
appear  to  be  rigid  and  straight  as  a  rule,  but  occasional  rods  are 
observed  to  be  slightly  curved.  In  fluid  media  there  is  a  tendency 
for  the  organisms  to  develop  long  tangled  filaments  in  which  granules 


FIG.  78. — Vincent's  angina,  Bacillus  and  Spirillum  fusiformis. 

may  be  absent.  Motility  has  not  been  observed,  and  spores  and  cap- 
sules have  never  been  demonstrated.  Ordinary  stains  color  the  organ- 
isms faintly,  but  stains  containing  mordants,  as  carbolfuchsin  and 
carbolthionin,  stain  them  readily  and  one  or  two  intensely  colored 
granules  are  frequently  observed  in  each  organism.  The  organisms 
are  Gram-negative. 

Tunnicliff7  obtained  development  of  fusiform  bacilli  in  ascitic  fluid 
media  (anaerobic)  at  37°  C.,  but  subcultures  were  usually  negative. 
Krumwiede  and  Pratt,8  using  an  improved  anaerobic  culture  method, 
obtained  pure  cultures  in  anaerobic  ascitic  agar  or  serum  agar  from 

1  Deutsch.  med.  Wchnschr.,  1894,  xlix,  922.  2  Ann.  Inst.  Past.,  1899,  609. 

3  Ibid.,  1896,  488.  4  Centralbl.  f.  Bakt.,  1898,  xxiii,  177. 

5  Brault,  Bull.  Derm,  et  Syph.,  1908,  2.  6  Jour.  Am.  Med.  Assn.,  1913,  Ix,  1769. 

7  Jour.  Inf.  Dis.,  1906,  iii,  148.  » Ibid.,  1913,  xii,  199;  xiii,  438. 


FUSIFORM  BACILLI  AND  SPIRILLUM  FUSIFORMIS       531 

a  variety  of  lesions  of  the  type  mentioned  above.  The  colonies  were 
small,  more  or  less  circular  in  outline  with  projecting,  hair-like  growths, 
which  attain  a  diameter  of  1  to  2  mm.  In  all,  fifteen  strains  were 
isolated  in  pure  culture,  all  of  which  produced  indol  and  possessed  a 
disagreeable  odor.  Two  distinct  cultural  types  were  distinguished; 
all  strains  produced  acid,  but  no  gas,  in  dextrose,  galactose  and  levu- 
lose;  one  type  produced  acid  in  saccharose,  the  other  type  was  with- 
out action  upon  this  sugar.  There  was  no  demonstrable  relation 
between  the  source  of  the  culture  and  the  fermentation  of  saccharose, 
which  is  in  harmony  with  Tunnicliff's  observation  that  the  fusiform 
bacilli  obtained  from  a  variety  of  lesions  presented  no  demonstrable 
distinctive  characters. 

No  spiral  organisms  developed  in  the  cultures,  although  they  were 
present  in  smears  from  the  original  material.  This  points  strongly 
to  the  non-identity  of  the  fusiform  bacillus  and  the  spiral  organism 
so  frequently  associated  with  it,  although  Tunniclift'1  claims  that  the 
spirilla  and  the  fusiform  bacilli  are  different  forms  of  a  single  organism. 

The  relation  of  the  fusiform  bacilli  to  morbid  processes  is  not  finally 
established,  although  the  injection  of  material  rich  in  these  organisms 
has  frequently  led  to  necrosis  and  suppuration  in  experimental  animals. 
The  most  convincing  evidence  of  their  pathogenicity  is  the  occasional 
demonstration  of  fusiform  bacilli  in  considerable  numbers  in  tissues 
from  cases  of  noma  and  similar  severe  lesions. 

1  Jour.  Inf.  Dis.,  1911,  viii,  316. 


SECTION  III. 

HIGHER  BACTERIA,  MOLDS,  YEASTS,  FILTERABLE 
VIRUSES,  DISEASES  OF  UNKNOWN  ETIOLOGY. 


CHAPTER  XXVIII. 

TRICHOMYCETES,  ACTINOMYCETES,   HYPHOMYCETES, 
SACCHAROMYCETES. 


THE  PATHOGENIC  HIGHER  BACTERIA. 
Trichomy  cetes . 
Leptothrix. 
Cladothrix. 
Nocardia  (Strep tothrix). 


Actinomyces  Bo  vis. 

Mycetoma  (Madura  Foot). 
HYPHOMYCETES. 

Eumycetes  or  Molds. 
SACCHAROMYCETES. 


THE   PATHOGENIC   HIGHER   BACTERIA. 

Trichomycetes.— The  Trichomy  cetes  occupy  a  position  intermediate 
between  the  true  bacteria  (Schizomycetes)  and  the  molds  (Hypho- 
my cetes),  in  the  system  of  classification.  Their  method  of  reproduc- 
tion is  more  complex  than  that  of  the  bacteria,  but  their  cycle  of 
development  is  simpler  than  that  of  the  molds.  The  organisms 
usually  grouped  in  the  Trichomycetes  are  heterogeneous  in  their 
characteristics  and  there  is  a  decided  lack  of  agreement  concerning 
the  limitation  of  the  several  subdivisions  of  these  microorganisms. 
Foulerton1  places  all  the  members  of  the  higher  bacteria  in  one  genus, 
Streptothrix,  including  the  older  genera,  Leptothrix,  Cladothrix, 
Streptothrix  and  Actinomyces.  Wright2  and  others  have  not  sub- 
scribed to  this  view  and  their  evidence  is  impressive.  Additional 
investigations  are  required  before  final  judgment  can  be  made.3  For  the 
present  the  older  grouping  of  the  Trichomycetes,  Leptothrix,  Clado- 
thrix, Nocardia  (Streptothrix),  and  Actinomyces  will  be  adhered  to. 

1Allbutt  and  Rolleston,  System  of  Medicine,   1906,  ii,  Part  I,  302;    British  Med. 
Jour.,  1912,  i,  300. 

2  Jour.  Med.  Research,  1905,  xiii,  349. 

3  See  Musgrave,  Clegg,  and  Polk,  Philippine  Jour,  of  Sci.,  1908,  iii,  447,  for  very  full 
bibliography  and  discussion. 


534      TRICHOMYCETES,  -ACTINOMYCETES,  HYPHOMYCETE8 

Leptothrix. — Leptothrices  are  frequently  found  in  the  mouth,  so 
commonly  indeed  that  Leptothrix  buccalis  is  regarded  as  a  regular 
inhabitant  of  the  oral  cavity.  Suppurative  processes  incited  by  this 
organism  have  been  reported  by  a  few  observers,  but  the  evidence 
is  by  no  means  conclusive.  The  organisms  are  cultured  with  great 
difficulty  upon  artificial  media  and  no  cultures  were  obtained  from 
the  cases  reported. 

Cladothrix. — The  important  cultural  differentiation  of  the  Clado- 
thrices  from  the  Streptothrices  rests  upon  the  false  branching  of  the 
former.  The  few  meager  reports  of  cases  of  Cladothrix  infection  cited 
in  the  literature  are  not  sufficiently  definite  to  determine  the  type  of 
organisms  involved. 


FIG.  79. — Streptothrix  hominis. 

Nocardia  (Streptothrix). — The  more  common  name  of  the  group  is 
Streptothrix,  but  investigation  has  shown  that  the  latter  term  was 
previously  given  to  a  mold;  according  to  rules  of  botanical  nomen- 
clature, it  must  be  replaced  by  a  name  hitherto  unused.  Nocardia 
appears  to  be  appropriate.  The  first  organism  was  described  by 
Nocard1  as  the  inciting  agent  of  a  disease  of  cattle  in  Guadaloupe, 
known  as  farcin.  Since  that  time  many  cases  have  been  reported 
both  in  animals  and  in  man. 

Nocardia  mycoses  have  occasionally  been  confusedlwith  tuberculous 
infections  in  the  past.  Farcin  was  suspected  to  be  a  tuberculous 
process  until  Nocard2  clearly  demonstrated  that  the  organism  was  an 
acid-fast  Nocardia. 

In  man  the  disease  usually  progresses  slowly  and  the  lesions  are 
markedly  localized,  but  it  may  run  a  rapidly  fatal  pyemic  or  pneu- 

1  Ann.  Inst.  Past.,  1888,  ii,  293.  2  Loc.  cit. 


THE  PATHOGENIC  HIGHER  BACTERIA  535 

monic  course  of  one  or  two  weeks'  duration.  A  chronic  case  may 
abruptly  become  generalized  and  terminate  fatally.  It  is  not  defi- 
nitely known  if  all  chronic  cases  prove  fatal  or  if  some  eventually 
recover.  The  Nocardia  appear  to  be  widely  distributed  in  the  soil, 
water,  upon  foodstuffs  and  upon  plants  and  it  is  suggestive  that 
nearly  50  per  cent,  of  all  cases  reported  have  been  infections  of  the 
head  and  neck.1  About  20  per  cent,  of  cases  are  chest  infections  and 
the  clinical  symptoms  are  very  like  those  of  tuberculosis.  If  repeated 
sputum  examinations  are  negative  although  the  syndrome  suggests 
tuberculosis,  search  should  be  made  for  Nocardia. 

Morphology. — The  Nocardia  are  very  pleiomorphic;  in  purulent 
material  and  other  discharges  the  organisms  are  of  varying  length, 
some  short  and  rod  shaped,  others  long-branched  filaments  (mycelia). 
The  filaments  usually  segment  or  fragment,  producing  the  shorter 
bacillary  forms  and,  in  artificial  media,  forming  chains  of  spores  as 
well.  Old  cultures  in  artificial  media  are  composed  chiefly  of  bacilloid 
forms — long,  somewhat  curved  filaments  which  may  or  may  not  be 
branched,  and  spores  which  occur  singly  or  in  small  groups  and  pairs. 
The  organisms  may  or  may  not  be  acid-fast,  but  they  are  Gram- 
positive.  The  granules  or  udrusen"  so  characteristic  of  actinomycotic 
infections  are  not  found  in  Nocardial  mycoses. 

Cultivation. — Nocardia  may  frequently  be  grown  upon  artificial 
media — gelatin  or  agar — directly  from  pus  or  other  morbid  material. 
The  colonies  develop  slowly  and  after  five  to  seven  days  they  appear 
as  gray,  opaque,  shining  plaques  which  may  reach  3  to  5  mm.  in 
diameter  after  prolonged  incubation.  A  densely  matted  pellicle 
composed  of  branched  and  unbranched  filaments  forms  upon  the 
surface  of  broth  and  a  flocculent  sediment  gradually  collects  at  the 
bottom  of  the  tube.  Loffler's  blood  serum  appears  to  be  the  most 
favorable  medium  for  the  initial  growth  of  Nocardia  directly  from  the 
tissues. 

The  inoculation  of  cultures  into  rabbits  or  guinea-pigs  frequently 
leads  to  chronic  abscesses,  bronchopneumonia  or  a  rapidly  fatal 
generalized  infection,  depending  upon  the  virulence  of  the  organism 
and  the  site  of  inoculation.  Recently  Claypole2  has  prepared  a  series 
of  "Streptotrichins;"  glycerin  bouillon  cultures  made  from  non-acid- 
fast  mycelial  organisms  and  the  partly  acid-fast  bacillary  forms  of 
Nocardia,  which  give  definite  skin  reactions  on  persons  with  nocardial 

1  Claypole,  Jour.  Am.  Med.  Assn.,  1914,  xiii,  604. 

2  Jour.  Am.  Med.  Assn.,  1914,  Ixiii,  603. 


536      TRICHOMYCETES,  ACTINOMYCETES,  HYPHOMYCETES 

infections.  Controls  (normal,  uninfected  individuals),  do  not  react, 
but  a  Nocardial  mycosis  and  tuberculosis  may  exist  simultaneously 
in  the  same  individual,  as  shown  by  the  appearance  of  both  organisms 
in  the  sputum,  and  both  the  streptotrichin  and  tuberculin  skin  reac- 
tions. Claypole  also  finds  that  glandular  and  bone  infections  with 
Nocardia  may  be  demonstrated  as  readily  as  the  lung  infections  by 
the  skin  reaction  with  streptotrichin. 

Actinomyces  Bovis. — Synonyms. — Discomyces  bovis;  Nocardia  acti- 
nomyces;  Streptothrix  israeli. 

The  causative  organism  of  the  disease  of  cattle  known  as  "lumpy 
jaw"  or  "big  jaw,"  Actinomyces  bovis,  was  first  described  by  Bol- 
linger,1  although  the  granules  or  "drusen,"  consisting  of  colonies  of 
the  organism,  were  described  by  von  Langenbeck  as  early  as  1845. 
The  first  human  cases  were  reported  by  Israel.2 


FIG.  80. — Actinomyces  colony  showing  peripherally  arranged  clubs. 

Considerable  confusion  has  arisen  concerning  the  identity  of  the 
organisms  found  in  suppurative  lesions  which  superficially  closely 
resemble  those  of  Actinomycosis.3  Wright4  has  clearly  shown  that 
true  actinomycotic  infections  are  characterized  not  only  by  suppura- 
tive processes  and  granulation  tissue  formation,  but  that  the  pus 
from  these  lesions  contains  the  characteristic  granules  or  "drusen," 
which  are  composed  of  branched  filamentous  organisms  densely  packed 
together,  with  characteristic  club-shaped  bodies  radially  arranged 

1  Centralbl.  f.  klin.  Med.  Wissensch.,  1877,  xv,  481. 

2  Virchows  Arch.,  1878,  Ixxiv,  15;    1879,  Ixxviii,  421. 

3  See  Foulerton  (Trans.  Path.  Soc.,  London,   1902,  liii,  Part  1,  56),  and  Neukirch 
(Ueber  Strahlenpilze,  Strassburg,   1902),  for  literature. 

4  Jour.  Med.  Res.,  1905,  xiii,  349. 


THE  PATHOGENIC  HIGHER  BACTERIA  537 

at  the  periphery  of  the  colony.  The  pus  from  so-called  pseudo- 
tuberculosis,  streptothrix  and  cladothrix  infections  do  not  exhibit 
these  characteristic  "drusen." 

Morphology. — Actinomyces  bovis  is  a  pleiomorphic  organism  belong- 
ing to  that  group  of  microorganisms  intermediate  between  the  true 
bacteria  (Schizomycetes)  and  the  molds  (Hyphomycetes)  known  as 
the  Trichomycetes.  It  is  best  observed  in  pus  from  active  lesions, 
in  which  it  occurs  in  gray  or  yellowish  colonies  or  granules  (drusen), 
frequently  large  enough  to  be  visible  to  the  naked  eye.  The  colonies 
vary  in  size  but  usually  measure  from  0.5  to  2  mm.  in  diameter. 
Such  a  colony,  crushed  between  two  slides  or  a  slide  and  cover  glass, 
appears  as  a  rosette-shaped  aggregation  of  densely  packed  filaments 


FIG.  81. — Actinomyces,  bouillon  culture. 

which  exhibit  a  radial  arrangement.  The  centre  is  so  crowded  with 
organisms  that  it  appears  opaque  and  usually  contains  many  ovoid 
bodies  measuring  from  1  to  1.5  microns  in  diameter.  According  to 
Wright,1  these  ovoid  or  coccoid  bodies  are  formed  by  the  disintegration 
of  the  filaments.  The  periphery  of  the  colony  contains  many  inter- 
laced branching  filaments,  many  of  which  exhibit  on  their  distal  ends, 
an  enlargement  or  "club"  which  is  a  hyaline  layer  or  sheath  about 
the  extremity  of  a  filament.  These  filaments  measure  about  10  to 
12  microns  in  length  and  the  clubs  20  to  30  microns  in  length  by  8 
to  10  microns  in  diameter.  Grown  in  artificial  media  club  formation 
is  absent  unless  blood  or  blood  serum2  is  added,  but  even  in  enriched 
media  the  formation  of  clubbed  forms  is  irregular. 

1  Loc.  cit.  2  Wright,  loc.  cit.,  p.  336. 


538      TRICHOMYCETES,  ACTINOMYCETES,  HYPHOMYCETES 

Actinomyces  bovis  stains  by  Gram's  method,  but  the  clubs  are  not 
colored.  Eosin  brings  them  out  clearly.  It  has  been  held  by  Bostrom1 
that  the  clubs  are  degenerative  phenomena,  but  Wright2  believes  their 
chief  function  is  a  protective  one,  shielding  the  filaments  from  the 
harmful  action  of  the  body  fluids  and  cells  of  the  host. 

Isolation  and  Culture. — The  organism  is  anaerobic  and  appears  to 
grow  with  moderate  luxuriance  in  deep  glucose-agar  stab  cultures. 
Material  for  inoculation  is  best  obtained  by  crushing  a  granule  between 
sterile  glass  slides,  or  rubbing  it  on  the  inside  of  a  sterile  test-tube, 
after  two  to  three  preliminary  washings  in  sterile  salt  solution  to 
remove  or  diminish  surface  contamination.  The  finely  macerated 
colony  is  distributed  evenly  in  deep  dextrose-agar  tubes  and  incubated 


FIG.  82. — Actinomyces — club  formation,  semi-diagrammatic. 


at  37°  C.  After  two  to  five  days  colonies  appear  scattered  through 
the  depths  of  the  medium  and  are  generally  very  numerous  in  a  zone 
0.5  to  1  cm.  below  the  surface.  They  do  not  ordinarily  grow  above 
this  level.  The  deeply  lying  colonies  increase  in  size  until  they 
measure  1  to  3  mm.  in  diameter  at  the  end  of  a  week's  incubation. 
Microscopically  these  colonies  consist  of  masses  of  radially  arranged, 
branching  filaments  which  exhibit  a  decided  tendency  to  break  up 
into  short  bacilloid  or  ovoid  segments.  A  colony  at  this  stage  becomes 
a  mass  of  compact  short  filaments  and  bacillary  forms.  Clubs  are  not 
seen  under  these  conditions  unless  blood  or  blood  serum  is  added  to 
the  medium. 

In  bouillon  the  organisms  grow  in  dense  white  or  gray  masses  of 


1  Beitr.  z.  path.  Anat.,  u.  z.  allg.  Path.,  1890,  ix,  1. 


2  Loc.  cit.,  p.  397. 


THE  PATHOGENIC  HIGHER  BACTERIA 


530 


interwoven  filaments  which  develop  only  at  the  bottom  of  the  tube. 
Surface  growth  is  never  observed  and  turbidity  practically  never 
occurs.  Freshly  heated  broth,  in  which  the  dissolved  oxygen  has 
been  driven  off,  appears  to  afford  a  somewhat  more  luxuriant  growth, 
particularly  during  the  first  few  days'  inoculation,  but  this  precau- 
tion is  by  no  means  absolutely  necessary  to  obtain  development. 
Prolonged  cultivation  in  broth  frequently  causes  the  organisms  to 
lose  the  discrete,  mulberry-like  colony;  the  growth  becomes  some- 
what flocculent  and  viscid.  Milk  and  other  artificial  media,  aside 
from  agar  and  bouillon,  are  not  favorable  for  the  development  of  the 
organisms. 


FIG.  83. — Actinomyces — mycelioid  development,  semi-diagrammatic. 


Actinomyces  bovis  does  not  grow  at  temperatures  much  below  37° 
C.  Development  ceases  at  room  temperature.  The  resistance  to 
drying  is  considerable,  fifty  days  being  about  the  minimal  time  required 
to  prevent  growth.  In  artificial  media,  however,  the  organism  usually 
becomes  non-viable  in  a  shorter  period.  The  thermal  death  point  is 
about  62°  C.  for  five  minutes.  Toward  ordinary  antiseptics,  Actino- 
myces is  very  resistant,  but  it  is  claimed  that  methylene  blue  is  strongly 
germicidal  to  it. 

Products  of  Growth. — Neither  toxins  nor  enzymes  have  been  detected 
in  cultures  of  Actinomyces  bovis.  It  is  believed  that  toxins  are  not 
produced. 

Pathogenesis. — Animal  and  Human. — Actinomycosis  occurs  as  a 
spontaneous  infection  both  in  cattle  and  in  man;  much  more  commonly, 
however,  in  the  former.  Other  mammals — horses,  asses  and  sheep — 
are  occasionally  infected.  The  lesions  belong  to  the  group  of  the 
infectious  granulomata  and  the  portal  of  entry  of  the  organism  is 


540      TRICHOMYCETES,  ACTINOMYCETES,  HYPHOMYCETES 

usually  the  mouth,  although  cutaneous  infections  have  been  described. 
The  mouth  and  adnexa  and  the  pharynx  are  more  commonly  the  site 
of  the  initial  localization  of  the  organism,  but  the  lungs  or  the  alimen- 
tary canal  may  be  first  involved.  The  earliest  stage  of  the  infection 
is  a  small  nodule  not  unlike  a  tubercle;  microscopically  it  is  made  up 
of  small  round  cells,  epithelioid  cells  and  giant  cells.  This  soon  softens 
and  sinuses  often  are  formed,  through  which  the  pus  escapes.  The 
surrounding  connective  tissue  proliferates  rapidly,  forming  a  dense 
encapsulation  through  which  invasion  of  neighboring  tissue  takes 
place;  often  the  disease  spreads  in  one  direction  wrhile  simultaneously 
the  older  lesion  becomes  cicatrized.  Death  frequently  occurs  through 
secondary  invasion  by  adventitious  bacteria. 


FIG.  84. — Mucor  sporangium, 

Actinomycosis  is  not  a  contagious  disease  and  it  is  practically 
impossible  to  infect  experimental  animals,  as  guinea-pigs  and  rabbits, 
with  the  virus.  Wright1  has  been  unable  to  produce  progressive 
actinomycosis  in  these  animals,  although  he  succeeded  occasionally 
in  inducing  a  localized  purulent  nodule  formation  in  guinea-pigs,  in 
which  granulation  tissues  and  colonies  of  Actinomyces  appeared,  some 
of  which  showed  poorly  defined  clubs. 

The  disease  is  stated  to  be  transmitted  through  wounds  caused  by 
certain  grains,  particularly  those  which  possess  barbs,  but  the  evidence 
is  not  wholly  convincing. 

The  diagnosis  of  actinomycosis  is  best  made  by  microscopic  exam- 
ination of  sputum,  or  the  pus  from  the  lesions.  The  demonstration 
of  the  characteristic  "drusen"  with  their  club-shaped  peripheral 

1  Loc.  cit. 


HYPHOMYCETES  541 

filaments  is  conclusive.  Sometimes  actinomycotic  pus  does  not 
contain  granules;  if  the  sinus  be  curetted,  the  organisms  will  fre- 
quently be  demonstrable  in  the  scrapings,  even  though  they  are  absent 
from  the  pus. 

Mycetoma  (Madura  Foot). — The  term  Mycetoma  is  a  generic  one, 
including  purulent  inflammations  of  the  foot  chiefly,  but  also  of  the 
hands  and  less  commonly  of  other  parts  of  the  body.  The  lesions 
superficially  resemble  those  of  actinomycosis. 

Three  varieties  of  the  disease  have  been  described,  depending  upon 
the  color  of  the  granules  found  in  the  pus — the  melanoid  or  black  type, 
the  ochroid  or  white,  and  a  red  type  which  has  been  less  thoroughly 
investigated. 

Several  organisms  have  been  isolated  from  the  various  lesions, 
including  not  only  an  Actinomyces  (Actinomyces  madurse),  but  a 
mold,  Aspergillus  bouffardi,  as  well. 

The  mutual  relations  of  the  organisms  and  the  various  types  of 
Madura  foot  have  not  been  satisfactorily  determined. 

HYPHOMYCETES. 

Eumycetes  or  Molds. — The  molds  are  a  group  of  organisms  which 
are  structurally  somewhat  more  complex  than  Bacteria  for,  with  a 
very  few  exceptions,  there  is  a  physiological  division  of  function  into 
vegetative  cells  which  provide  the  nutrition  of  the  organism  and 
reproductive  cells  which  are  concerned  in  the  perpetuation  and  mul- 
tiplication of  the  species.  They  are  widely  distributed  in  nature, 
the  majority  living  saprophytically  upon  lifeless  organic  matter — 
some  are  parasitic  upon  animals  and  plants;  few  types,  however, 
incite  disease  in  man,  animals,  or  plants. 

In  human  pathogenesis  their  activities  are  usually  restricted  to  the 
skin  and  adnexa,  but  occasionally  spreading  over  mucous  membranes 
and  even  involving  the  respiratory  tract.  Among  the  hyphomyceal 
diseases  of  man  are  favus,  ringworm,  thrush,  pityriasis  versicolor, 
sporotrichosis  and  aspergillosis. 

The  cells  of  molds  are  larger  than  bacteria,  as  a  rule,  measuring 
on  the  average  from  2  to  10  microns  in  diameter,  and  they  grow  into 
long  filaments  or  threads  called  hyphae,  which  tend  to  branch  and  form 
intricately  interwoven  networks  called  mycelia.  Like  all  true  plant 
cells,  each  hypha  exhibits  a  clearly  defined,  doubly  contoured  ecto- 
plasm or  limiting  membrane  within  which  is  confined  the  cytoplasm, 


542      TRICHOMYCETES,   ACTINOMYCETES,   HYPHOMYCETES 

which  is  usually  coarsely  or  finely  granular.  In  the  lower  forms, 
Phycomycetes,  each  hypha  is  a  unicellular  multinuclear  cell,  which 
may  be  branched;  in  the  higher  forms,  My  corny  cetes,  the  filaments 
are  multicellular,  each  cell  being  separated  from  its  fellows  by  distinct 
septa.  A  nucleus  is  demonstrable  in  a  majority  of  the  molds  and  it 
is  probable  that  it  is  present  in  all. 

Reproduction. — The  reproductive  cells  of  the  lowest  and  simplest 
forms  are  scarcely  differentiated  morphologically  from  the  vegetative 
cells,  indeed  in  some  instances  the  distinction  has  never  been  made. 
The  hyphse  break  up  and  the  fragments  give  rise  to  new  colonies. 
Reproduction  in  the  Phycomycetes,  of  which  the  widely  distributed 
genus  Mucor  is  a  familiar  type,  occurs  in  the  following  manner — a 
constriction  occurs  near  the  tip  of  an  aerial  hypha  and  the  extremity 


FIG.  85. — Aspergillus  sporangia. 

then  increases  in  size  until  a  spherical  mass,  the  sporangium,  is  formed, 
which  divides  into  a  number  of  spores.  These  escape  with  the  rupture 
of  the  sporangium  and,  if  they  reach  a  favorable  medium,  form  the 
starting  points  of  new  colonies.  This  is  asexual  reproduction.  Sexual 
reproduction  takes  place  somewhat  differently:  lateral  branches 
from  two  adjacent  hyphse  meet  and  fuse.  These  branches  or  gameto- 
phores  are  morphologically  indistinguishable  but  differ  in  sex.  The 
fused  cell  enlarges  to  form  a  zygospore,  separated  from  the  hyphse 
by  septa,  and  eventually  grows  into  a  sporangium,  from  which  asexual 
spores  escape  and  start  new  colonies. 

Among  the  Mycomycetes  or  higher  molds,  asexual  reproduction  alone 
occurs.  The  simplest  type  begins  as  a  thickening  of  the  end  of  a  hypha, 
which  soon  constricts  at  regular  intervals  to  form  small  spherical  or 


HYPHOMYCETES  543 

oval  spores.  The  spore-containing  cell  is  known  as  an  ascus  and  the 
spore  ascospore. 

Somewhat  more  complex  is  reproduction  of  the  common  green 
mold,  Aspergillus.  An  aerial  hypha  or  conidiophore  develops,  thicker 
at  the  distal  than  at  the  proximal  end,  and  from  this  thickened  end 
radially  arranged  spherical  or  oval  conidia  arise. 

Microscopical  Examination  of  Molds.  —  Molds  are  usually  best 
examined  in  water,  to  which  an  equal  volume  of  glycerin  has  been 
added,  and  unstained.  The  general  arrangement  of  mycelium,  spores 
and  sexual  bodies  can  be  observed  with  the  lower  powers  of  the 
microscope — the  finer  details  of  structure  require  a  greater  magni- 
fication. Anilin  dyes  color  molds  readily  and  the  Weigert  fibrin- 
staining  method  is  very  good  to  demonstrate  molds  in  tissue  sections. 


FIG.  86. — Penicillium;  conidiophores,  sterigmata,  and  conidia. 

Growth  on  Artificial  Media. — Molds  are  almost  invariably  aerobic 
and  their  development  in  artificial  media  requires  abundant  free 
oxygen.  A  slightly  acid  reaction  is  best  for  their  growth,  but  media 
with  an  alkaline  reaction  and  even  a  relatively  strong  acid  reaction 
(organic  acids,  not  mineral  acids),  will  usually  permit  of  their  mul- 
tiplication. Even  on  very  dry  media  development  takes  place. 

Pathogenic  Molds. — Favus. — Favus  or  tinea  favosa  is  a  skin  disease 
limited  chiefly  to  the  hairy  parts  of  the  body;  more  frequently  the 
head  alone  is  involved,  but  the  disease  may  spread  over  the  entire 
surface  of  the  body.  It  is  not  limited  to  man — dogs,  cats,  mice  and 
rabbits  are  also  susceptible.  The  disease  is  contagious  and  is  trans- 
mitted from  man  to  man  or  from  animal  to  man  by  contact.  Unclean- 
liness  is  a  potent  predisposing  factor,  but  individuals  with  lowered 


544      TRICHOMYCETES,  ACTINOMYCETES,   HYPHOMYCETES 

vitality,  as  poorly  nourished  children  and  consumptives,  appear  to 
be  relatively  more  readily  infected  than  the  more  robust.  The  organ- 
ism spreads  slowly  and  the  disease  is  a  chronic  one,  difficult  to  influence 
by  treatment.  The  initial  lesions  are  small  red  pimples,  which  soon 
enlarge  somewhat,  forming  gray  or  sulphur-yellow  crusts  grouped 
around  the  base  of  hairs/  These  crusts,  known  as  scutella  (singular 
scutellum),  slowly  increase  in  size  peripherally  and  tend  to  coalesce. 
If  a  scutellum  is  removed  it  is  found  to  be  somewhat  thicker  in  the 
centre  and  cup  shaped.  Examined  under  the  microscope  it  consists 
of  a  dense,  matted  mycelium  which  in  the  centre  may  be  so  compact 
as  to  obscure  the  individuality  of  the  filaments ;  at  the  periphery  the 
growth  is  less  luxuriant  and  the  individual  filaments  are  clearly 
defined.  Spores  are  very  numerous  at  the  centre  of  the  scutellum,  but 
at  the  periphery  they  are  much  fewer  in  numbers.  The  hair  enclosed 
by  the  colony  of  mold  is  destroyed. 

The  organism,  Achorion  schonleinii,  was  first  observed  by  Schonlein 
in  1839.  It  is  readily  cultivated  at  room  temperature  upon  gelatin, 
or  better,  upon  agar  at  30°  to  35°  C.  Media  with  a  neutral  or  slightly 
alkaline  reaction  are  more  favorable  for  its  development  than  acid 
media.  In  this  respect  Achorion  schonleinii  differs  culturally  from 
the  majority  of  molds.  Material  taken  directly  from  the  centre  of 
a  scutellum,  streaked  upon  agar,  usually  develops  into  white  or  gray 
colonies  in  which  the  mycelia  and  spores  are  readily  recognizable  with 
the  lower  powers  of  the  microscope.  Frequently  adventitious  organ- 
isms overgrow  the  more  slowly  developing  favus  parasite.  If  a  piece 
of  the  scutellum  is  ground  in  a  sterile  mortar  with  sterile  powdered 
water  glass  and  the  powder  well  distributed  upon  gelatin-agar  or 
Sabouraud's  medium,1  pure  cultures  are  usually  obtained.  The  yellow- 
brown  colony  usually  exhibits  a  central  depression  resembling  some- 
what that  of  the  scutellum.  The  swollen  ends  of  the  filaments  are 
quite  characteristic.  There  appear  to  be  several  varieties  of  the  mold, 
but  there  is  only  one  type  of  the  disease. 

Herpes  Tonsurans. — Herpes  tonsurans,  ringworm,  Tinea  tonsurans 
or  sycosis  is  a  disease  chiefly  of  the  hairs  of  the  head  or  beard,  but  it 
often  spreads  to  the  skin  as  well,  Tinea  circinata.  The  axillary  or 
pubic  hairs  are  occasionally  involved.  It  occurs  in  children  rather 

1  SABOURAUD'S   MEDIUM. 

Peptone  (Witte)         2.0  grams 

Glycerine,  redistilled,  pure 4.0  grams 

Water 100.0  c.c. 

Agar     ......            1.2  grams 


HYPHOMYCETES  545 

more  frequently  than  in  adults.  The  disease  is  characterized  clinically 
by  the  formation  of  inflamed  scab-areas  or  patches  on  the  skin  imme- 
diately surrounding  hairs  and  these  patches  exhibit  a  decided  tendency 
to  spread.  They  itch  intensely  and  within  them  the  hairs  fall  out. 
Usually  the  inflammation  is  not  accompanied  by  exudation,  but  in 
very  severe  cases  pustule  formation  may  occur.  The  disease  is  con- 
tagious and  is  transmitted  by  towels,  the  hands,  hairdressers'  utensils 
and  very  commonly  in  the  tropics  through  laundry.  The  initial  lesion 
appears  in  the  outer  layers  of  the  skin  and  extends  downward  through 
the  hair  follicle  and  then  invades  the  inner  layers  of  the  hair  itself, 
through  which  both  the  mycelia  and  spores  develop  in  large  numbers. 

The  organism,  Trichophyton  tonsurans,  was  described  by  Gruby 
and  by  Malmsten  in  1845.  Several  subvarieties  have  been  described, 
but  their  differential  characteristics  are  imperfectly  established.  It 
is  readily  demonstrated  in  the  hair  bulb  by  adding  a  few  drops  of 
NaOII  solution,  gently  heating  and  examining  under  the  microscope.1 
The  mycelial  filaments  appear  in  the  bulb  and  penetrate  for  some 
distance  along  the  hair  shaft.  The  spores  are  usually  restricted  to 
the  outer  layers  of  the  hair. 

The  mold  grows  readily  upon  neutral  agar  and  gelatin,  the  latter 
becoming  liquefied.  After  a  few  days'  incubation,  multicellular 
mycelia  with  their  nodal  thickenings  within  which  chlamydospores 
develop  appear  and  frequently  the  colony  becomes  pigmented  — 
brownish — after  prolonged  cultivation.  Plant2  states  that  there  are 
two  varieties  of  Trichophyton  tonsurans,  one,  less  common,  produc- 
ing relatively  large  spores,  the  other  producing  smaller  spores.  Guinea- 
pigs  may  be  successfully  infected  with  cultures  of  the  organisms 
grown  on  artificial  media;  a  small  area  on  the  back  is  epilated  and  the 
culture  rubbed  in.  The  lesions  are  self-limited  and  usually  heal 
spontaneously  after  a  few  weeks. 

Pityriasis  Versicolor. — Pityriasis  versicolor  is  a  disease  of  .the 
epidermis  which  differs  from  favus  and  ringworm  anatomically  in 
that  the  infecting  organisms  neither  penetrate  the  deeper  layers  of 
the  skin,  nor  do  they  cause  any  noteworthy  alterations  in  the  skin  or 
hair.  Usually  the  epidermis  of  the  chest,  abdomen,  joints  and  axilla 
are  involved,  rarely  the  neck.  The  disease  is  observed  in  the  uncleanly 

1  Water  should  not  be  added  after  the  addition  of  the  NaOH,  else  the  hair  will  very 
quickly  crumble. 

2  Plant  removes  a  hair  to  a  sterile  moist  chamber,  seals  the  cover  glass  with  melted 
paraffin  and  incubates  for  several  days.     When  the  spores  have  germinated  the  mycelia 
may  be  removed  and  cultivated  upon  agar  or  gelatin. 

35 


546      TRICHOMYCETES,  ACTINOMYCETES,  HYPHOMYCETES 

and  particularly  in  those  who  prespire  freely.  The  tuberculous  and 
diabetics  are  not  infrequently  infected.  The  disease  is  characterized 
by  the  development  of  light  brown  or  yellow  patches  which  are  not 
noticeably  raised  above  the  surrounding  surface;  these  patches  are 
irregular  in  outline  and  tend  to  spread  and  coalesce. 

The  inciting  organism,  Microsporon  furfur,  was  described  in  1846 
by  Eichstedt.  The  organism  resembles  the  Achorion  schonleinii 
rather  closely.  It  occurs  in  abundance  in  the  scales  where  the  relatively 
short,  thick,  septate  hyphse  surrounded  by  large  groups  of  spores 
are  quite  characteristic.  The  hyphse  measure  from  3  to  4  microns 
in  diameter  and  the  spores  are  frequently  observed  to  be  enclosed  in 
a  spirally  coiled  covering.  They  measure  about  3  to  6  microns  in 
diameter. 

Cultivation  of  the  organism  upon  artificial  media  is  accomplished 
with  difficulty  and  glycerin  media  are  best  adapted  for  this  purpose. 
The  colonies  are  very  minute — 0.5  to  1  mm.  in  diameter.  They  are 
white  or  brownish  and  tend  to  spread  over  the  medium.  The  hyphae 
are  usually  definitely  curved  and  the  ends  are  somewhat  club  shaped. 
The  spores  occur  in  masses  very  similar  in  arrangement  to  those 
observed  in  the  scale  itself.  Cultures  rubbed  into  an  epilated  area  on 
the  back  of  rabbits  may  induce  the  characteristic  colored  patches  if 
the  inoculated  area  is  protected  with  a  thick  covering  to  induce 
hyperemia. 

Thrush  or  Soor. — Thrush  is  primarily  a  localized  disease  of  the 
mouth,  occurring  chiefly  in  weakly  children.  It  has  also  been  found 
in  the  vagina  of  pregnant  women  and  in  adults  suffering  from  severe 
nutritional  disturbances,  diabetes  and  typhoid  fever.  The  early 
lesion  is  a  small  white  plaque  which  has  a  velvety  appearance,  differing 
in  this  respect  from  the  pseudomembrane  of  diphtheria  and  from  the 
gray  throat  of  scorbutus.  The  plaque  is  made  up  of  epithelial  cells 
overgrown  with  the  organism.  The  lesion  may  spread  to  the  larynx 
and  esophagus  and  lead  to  a  generalized  fatal  infection.  Usually, 
however,  the  prognosis  is  favorable. 

The  organism,  Oidium  albicans,  was  described  by  Langenbeck  in 
1839,  but  it  was  first  successfully  cultured  by  Grawitz  in  1871.  The 
classification  of  Oidium  albicans  is  not  clear,  for  the  organism  grows 
both  as  a  yeast  and  produces  mycelia  and  spores.  The  yeast-like 
cells  are  oval  or  round,  measuring  about  4  to  6  microns  in  diameter, 
and  they  frequently  form  buds  precisely  like  true  yeasts.  They 
stain  mahogany  brown  with  strong  Gram's  solution.  The  mycelia 


HYPHOMYCETES  547 

are  doubly  contoured  and  form  chlamydospores.  If  a  bit  of  the 
membrane  be  macerated  in  a  drop  of  acetic  acid  the  epithelial  cells 
are  cleared  and  the  parasite  is  readily  observed.  Two  distinct  types 
are  recognizable  in  gelatin  cultures,  one  of  which  liquefies  the  medium, 
the  other  does  not.  In  solid  media  yeast-cell  formation  predominates 
and  many  of  the  cells  are  observed  to  bud;  in  fluid  media  mycelia 
are  produced  and  spore-formation  usually  occurs  after  several  days' 
incubation.  The  spores — chlamydospores — usually  enlarge  and 
develop  into  filaments  when  they  are  transplanted  into  fresh  media. 
The  organism  is  not  uncommon  in  the  air. 

The  organism  does  not  produce  thrush  when  introduced  into 
experimental  animals,  but  it  may  cause  a  generalized  thrush  mycosis 
when  injected  intravenously  in  rabbits. 


FIG.  87. — Spqrothrix. 

Aspergillus  Mycosis. — Aspergillus  fumigatus  occasionally  incites 
a  disease  of  the  lungs  and  bronchi  in  birds  and  rarely  in  man.  The 
organism  penetrates  to  the  alveoli  and  the  mycelia  and  spores  may 
be  demonstrated  in  sections  of  the  lungs  in  fatal  cases.  It  also  has 
been  found  rarely  in  middle-ear  infections  and  in  the  nasopharynx. 

The  mold  grows  readily  upon  ordinary  media  and  the  colonies, 
after  several  days,  become  dark  green  in  color.  The  organism  belongs 
to  the  genus  Aspergillus,  which  is  widely  distributed  in  damp  cellars 
and  upon  food.  Microscopically,  aerial  hyphse  arise  from  the  fila- 
mentous mycelium,  whose  distal  ends  are  swollen  into  club-shape 
masses  of  undivided  sterigmata,  from  which  chains  of  conidia  arise. 
The  conidia  are  spherical,  greenish,  and  measure  about  3  microns  in 
diameter.  It  is  differentiated  from  many  of  the  aspergilli  by  its  green 


548      TRICHOMYCETES,  ACTINOMYCETES,  HYPHOMYCETES 

color,  other  members  of  the  group  exhibiting  black,  brown  and  other 
colored  colonies. 

Rabbits,  guinea-pigs  and  pigeons  are  susceptible  to  infection  with 
Aspergillus  fumigatus.  The  lesions  produced  resemble  tubercles  some- 
what on  superficial  examination,  but  microscopic  examination  always 
reveals  the  mycelium  and  spores. 

Sporotrichosis. — The  disease  known  as  sporotrichosis  was  first 
described  by  Schenck1  and  later  by  Hektoen  and  Perkins.2  The 
latter  observers  named  the  causative  organism  Sporothrix  (Sporo- 
trichon)  schencki. 

Usually  sporotrichosis  runs  a  chronic  course,  characterized  by 
small  discrete  nodules  in  the  subcutis,  which  at  first  are  hard  and 
inelastic,  indolent  and  resemble  multiple  disseminated  gummata.  The 
lesions  progress  slowly  and  after  some  time  soften,  break  through  the 
skin  and  discharge  a  slimy,  serous,  yellowish  pus.  The  skin  around 
the  nodules  is  not  usually  greatly  indurated  and  there  is  little  pain, 
febrile  reaction  or  constitutional  disturbance.  Not  infrequently 
regional  lymph  channels  become  ^thickened  with  a  few  gumma-like 
nodules  at  irregular  intervals,  which  break  down  and  ulcerate.  The 
lesions  resemble  syphilitic  gummata,  or,  occasionally,  tuberculous 
ulcerations.  Rarely  the  disease  may  be  acute  with  fever,  emaciation 
and  prostration  and  sporotrichic  nodules  form  on  mucous  surfaces 
in  the  peritoneum,  the  lungs  or  kidneys.  The  Wassermann  reaction 
is  negative  and  neither  Treponemata  nor  tubercle  bacilli  are  found  in 
uncomplicated  cases. 

The  organism  develops  readily  upon  ordinary  culture  media  which 
have  an  acid  reaction.  Material  for  inoculation  is  best  obtained 
from  a  softened  but  unopened  nodule.  The  colonies  grow  slowly  as 
small  plaques  which  develop  into  white  fluffy  masses  that  become 
brown  after  prolonged  cultivation.  Secondary  transfers  to  artificial 
media  develop  much  more  rapidly.  Many  strains  grow  better  at 
room  than  at  body  temperature. 

The  organism  as  seen  in  the  pus  consists  almost  exclusively  of 
oval  spores  measuring  from  2  to  4  microns  in  diameter  and  from  3  to 
6  microns  in  length;  they  are  frequently  collected  in  groups  or  masses 
of  from  3  to  30  or  more,  at  the  ends  of  the  filaments.  They  are  Gram- 
positive.  The  mycelia  are  found  in  cultures  as  filaments  about  2 
microns  in  diameter  and  from  20  to  40  microns  long. 

1  Johns  Hopkins  Hosp.  Bull.,  1898,  ix,  286. 

2  Jour.  Exp.  Med.,  1900,  v,  77. 


SACCHAROMYCETES  549 

Rats  are  quite  susceptible  to  inoculation  with  pus  from  lesions 
or  from  cultures.  The  disease  may  follow  an  acute  or  a  chronic  course, 
but  the  cutaneous  nodules  are  not  regularly  produced  in  this  animal 
— otherwise  the  lesions  are  fairly  typical.  In  the  acute  disease  the 
animal  usually  dies  within  two  weeks,  frequently  in  consequence  of  a 
degeneration  of  the  parenchyma  of  the  kidney.  The  organism  may  be 
recovered  from  the  blood  stream  or  the  kidneys — a  true  sporotrichon 
septicemia.  In  the  chronic  type  of  the  disease  the  mold  localizes  and 
results  in  the  formation  of  multiple  abscesses  in  the  internal  organs 
and  especially  in  the  testes.  Intraperitoneal  injections  usually  lead 
to  the  appearance  of  small  nodules  in  the  testes  and  internal  organs 
which  may  remain  discrete  or  become  confluent,  with  central  necrosis 
and  suppuration.  They  resemble  miliary  tubercles  superficially. 
Microscopically  the  relatively  large  oval  spores,  but  not  the  mycelia 
are  found.  The  disease  appears  to  occur  spontaneously  in  rats,  espe- 
cially the  testicular  type. 

The  serum  of  cases  of  sporotrichosis  frequently  agglutinates  the 
spores  of  the  organism  (best  obtained  by  grinding  cultures  to  dryness 
in  a  sterile  mortar,  then  diluting  with  salt  solution  and  filtering 
through  filter  paper)  in  dilution  from  1  to  200  even  1  to  1000.  The 
sera  of  normal  individuals  possesses  no  agglutinating  power  for  the 
organism.  Actinomycotic  serum  may  agglutinate  with  the  organism 
in  dilutions  as  great  as  1  to  50,  suggesting  common  group  agglutinins 
for  both  organisms.  Complement  fixation  is  apparently  not  specific. 

SACCHAROMYCETES. 

The  Saccharomycetes  or  yeasts  are  especially  characterized  by  their 
method  of  multiplication.  Unlike  the  Bacteriacese,  which  reproduce 
by  transverse  fission,  the  resulting  cells  being  of  equal  size,  the  yeasts 
reproduce  by  budding.  A  yeast  cell  about  to  reproduce  sends  out  an 
evagination  or  bud,  which  is  first  visible  as  a  minute  enlargement  on 
the  surface  of  the  parent  organism.  This  gradually  increases  in  size, 
still  maintaining  an  ovoid  shape  and  remaining  adherent  by  a  small 
isthmus  until  it  reaches  approximately  the  size  of  the  original  cell. 
The  isthmus  then  is  broken,  continuity  between  the  two  cells  is  inter- 
rupted and  the  fully  mature  individual  reproduces  in  like  manner. 
It  is  not  uncommon  to  find  budding  in  the  daughter  cell  before  it 
severs  its  connection  with  the  mother  cell,  if  the  environmental  con- 
ditions are  favorable  for  rapid  growth.  Many  yeasts  form  highly 


550      TRICHOMYCETES,  ACTINOMYCETES,  HYPHOMYCETES 

refractile  bodies — ascopores — within  their  cytoplasm  when  inviron- 
mental  conditions  become  unfavorable  for  further  development  and, 
unlike  the  bacteria,  each  yeast  commonly  produces  more  than  one 
spore,  usually  two,  three  or  four,  but  rarely  or  never  more  than  four. 
The  ascospore  is  outlined  by  a  doubly  contoured  membrane  and 
usually  it  remains  within  the  intact  maternal  cell.  At  sporulation 
each  ascospore  develops  into  a  mature  yeast  cell,  consequently 
sporulation  in  this  group  is,  in  a  sense,  a  process  of  reproduction,  for 
each  ascospore  is  potentially  equivalent  to  a  bud  in  that  it  develops 
into  a  complete  vegetative  cell. 

The   yeasts   are   of   considerable   importance   commercially;  some 
varieties  are  extensively  used  in  the  fermentation  of  malt  and  others 


FIG.  88. — Yeast  cells  showing  budding. 

are  employed  in  the  manufacture  of  bread.  In  either  case  the  organism 
liberates  carbon  dioxide  from  carbohydrates,  and  alcohol  as  well. 
This  activity  is  brought  about  by  an  intracellular  enzyme,  "zymase," 
which  may  be  obtained  in  an  active  state,  free  from  yeast  cells,  by 
crushing  the  latter  with  hydraulic  presses  and  filtering  off  residual 
cells  through  porcelain  filters.  Little  or  no  acid  is  formed  and  the 
yeast  fermentations  are,  in  general,  different  in  this  respect  from 
bacterial  fermentations  in  which  acid  formation,  but  not  alcohol 
formation,  is  the  rule. 

Structurally,  yeasts  exhibit  greater  complexity  than  the  bacteria. 
The  cytoplasm  of  the  yeast  cell  usually  exhibits  a  granular  or  vacuo- 
lated  appearance  and  nuclear  material,  or  at  least  structures  that 
color  like  nuclei  have  been  demonstrated. 

The  view  was  formerly  held  that  yeasts  had  some  etiological  rela- 


SACCHAROMYCETES 


551 


tionship  to  cancer.  Sanfelice1  and  others  have  cultivated  organisms 
closely  resembling  Blastomycetes  from  cancerous  tissue  and  have 
attempted  to  harmonize  the  appearance  of  the  yeasts  with  certain 
inclusion  bodies  within  cancer  cells.'  The  consensus  of  opinion  at  the 
present  time  is  wholly  against  this  hypothesis. 

Certain  varieties  of  yeast  are  definitely  known  to  incite  disease  in 
man  and  animals.  Busse2  isolated  a  yeast  which  he  called  Saccharo- 
myces  hominis  from  a  fatal  infection  in  a  woman  which  began  in  a 
tibial  abscess  and  somewhat  later  Gilchrist3  reported  a  case  of  blasto- 
mycetic  dermatitis  in  man.  Since  that  time  numerous  similar  cases 
have  been  recorded,  a  majority  of  them  around  Chicago.4  The  causa- 
tive organism  (Blastomyces),  has  been  variously  grouped  with  the 
yeasts  and  with  the  oidia.  It  is  usually  referred  to  as  a  yeast. 


FIG.  89. — Blastomyces — section  of  lung. 

Morphology. — Blastomycetes,  as  found  in  the  tissues,  are  ovoid 
or  spherical  cells  measuring  from  3  to  30  microns  in  diameter,  the 
smaller  dimension  being  the  more  common.  Mycelial  and  hyphaeal 
forms  are  found  in  cultures,  but  they  are  rarely  met  with  in  the  tissues. 
The  mycelial  filaments  measure  from  5  to  10  microns  in  diameter. 
The  cells  usually  occur  in  groups  of  fifteen  or  twenty  or  even  more, 
but  occasionally  single  organisms  are  met  with.  The  variation  in 
size  within  large  groups  of  Blastomyces  is  usually  very  considerable. 
A  thick  membrane  or  capsule  is  frequently  found  around  mature 
cells  within  the  tissues  of  the  body,  but  ascospores  have  not  been 
definitely  demonstrated.  The  Blastomyces  stain  with  ordinary 


1  Centralbl.  f.  Bakt.,  Orig.,  1902,  xxxi,  254. 

3  Johns  Hopkins  Hosp.  Rep.,  1896,  i,  296. 

4  See  Arch.  Int.  Med.,  1914,  xiii,  No.  4,  for  Case  Reports. 


2  Ibid.,  1894,  xvi,  175. 


552      TRICHOMYCETES,  ACTINOMYCETES,  HYPHOMYCETES       / 

anilin  dyes  and  they  are  Gram-negative.  They  are  best  observed 
unstained  in  hanging  drop  preparations  previously  treated  with 
NaOH,  which  brings  out  their  outline  sharply,  also  the  refractile 
layers  of  the  cell  membrane.  Of  particular  importance  is  the  recog- 
nition of  budding,  which  at  once  distinguishes  the  organisms.  The 
cytoplasm  is  granular  while  the  cell  as  a  whole  possesses  no  flagella 
and  is  consequently  non-motile;  the  granules  frequently  exhibit 
Brownian  movement. 

Isolation    and    Culture. — The    Blastomycetes   grow    with   moderate 
luxuriance  upon  Loffler's  blood  serum  and  glucose  agar.    Initial  pure 


FIG.  90. — Blastomyces,  maltose  broth  culture. 

cultures  are  somewhat  difficult  to  obtain,  however,  chiefly  because 
adventitious  organisms  are  almost  always  present,  which  overgrow 
the  more  slowly  developing  Blastomycetes.  It  is  necessary  to  dilute 
material  containing  the  organisms  with  sterile  salt  solution  or  broth 
and  to  crush  the  tissue  into  minute  fragments.  Once  pure  colonies 
are  obtained,  their  perpetuation  by  subculturing  is  readily  accom- 
plished. Slightly  .  acid  maltose  agar,  according  to  Ricketts,1  is  an 
excellent  medium  both  for  isolation  and  subsequent  cultivation.  The 
colonies  upon  solid  media  are  at  first  small,  white,  elevated  plaques 
which  later  become  gray  or  brownish.  After  a  few  days  the  growth 
becomes  wrinkled  and  the  mycelial  threads  and  aerial  hyphae  develop, 
which  gives  the  culture  a  moldy  appearance.  The  hyphse  fill  the 
tube  around  the  colony.  In  fluid  media  the  growth  at  first  is  a  floc- 
culent  mass  which  collects  at  the  bottom  of  the  tube;  a  membrane 
or  pellicle  usually  develops  on  the  surface  of  the  medium,  falls  to  the 

1  Jour.  Med.  Research,  1901,  vi,  377. 


SACCHAROMYCETES  553 

bottom,  and  a  new  membrane  forms.  A  moderate  growth  develops 
in  gelatin,  but  the  medium  is  not  liquefied.  A  slight  acidity,  but  no 
other  visible  change,  develops  in  milk  cultures. 

The  organism  is  strongly  aerobic  and  grows  at  room  or  body  tem- 
perature. At  the  lower  temperature  hyphse  are  more  freely  formed; 
at  the  higher  temperature  the  typical  budding  predominates  and  few 
or  no  hyphse  appear  until  after  several  days'  incubation.  Freezing 
does  not  kill  Blastomycetes,  but  an  exposure  to  60°  C.  for  five  minutes 
is  fatal  to  them. 

Products  of  Growth. — The  fermentation  reactions  are  variable. 
Some  strains  fail  to  ferment  dextrose  or  maltose,  while  others  produce 
gas  (CO2)  in  this  medium.  On  the  whole,  the  fermentative  powers 
of  the  Blastomycetes  are  much  less  than  those  of  the  saprophytic 
yeasts.  Toxins  and  enzymes  have  not  been  detected  in  cultures  of 
the  organisms. 

Pathogenesis. —  Human. — The  initial  lesion,  usually  cutaneous,  is 
a  papule  surrounded  by  an  area  of  hyperemia,  which  soon  becomes  a 
pustule  yielding  a  tenacious  pus.  The  ulceration  spreads  slowly,  dis- 
charging small  amounts  of  thick,  purulent  material  and  surrounded 
by  a  red  areola  in  which  numerous  papules  are  frequently  detectable. 
As  the  lesion  spreads  the  older  portions  of  the  lesion  tend  to  become 
cicatrized  and  to  heal.  The  progress  of  the  disease  is  very  slow,  fre- 
quently requiring  years  to  cover  an  area  of  a  few  square  inches.  It 
does  not  often  spread  to  mucous  surfaces,  but  occasionally  metastases 
occur  in  the  lungs.  According  to  Stober,1  involvement  of  bones  and 
metastatic  foci  in  the  spleen,  liver  and  kidney  have  been  observed  in 
a  few  cases. 

Animal  Experimentation. — Attempts  to  reproduce  blastomycetic 
infections  in  dogs,  rabbits,  guinea-pigs,  white  rats  and  mice  have 
been  unsuccessful  when  artificially  cultivated  organisms  from  human 
lesions  have  been  inoculated,  although  Klein2  isolated  a  blastomycete 
from  milk,  which  produced  gelatinous,  tumor-like  swellings  and 
glandular  enlargement  when  injected  subcutaneously  into  guinea- 
pigs.  Intraperitoneal  injections^  resulted  in  the  formation  of  firm 
nodules  in  the  liver,  lungs,  pancreas,  testes,  ovaries  and  intestines. 
The  nodules  were  composed  chiefly  of  masses  of  the  organisms.  Toki- 
shige3  and  Tartakowsky4  have  isolated  organisms  belonging  to  the 

1  Arch.  Int.  Med.,  1914,  xiii,  509.  2  Brit,  Med.  Jour.,  1901,  ii,  1. 

3  Centralbl.  f.  Bakt.,  1896,  xix,  105. 

4  Die  afrikanische  Rotz  der  Pferde,  St.  Petersburg,  1897. 


554      TRICHOMYCETES,  ACTINOMYCETES,  HYPHOMYCETES 

Blastomycetes  Group  from  a  cutaneous  infection  of  horses,  and  San- 
felice1  recovered  a  similar  organism  from  a  lymph  gland  of  an  ox  which 
had  a  generalized  carcinoma.  This  organism  was  pathogenic  for 
white  rats,  rabbits,  guinea-pigs,  sheep  and  cattle. 

The  defensive  mechanism  which  tends  to  limit  the  spread  of  the 
organism  in  the  body  is  largely  phagocytic,  together  with  a  prolifera- 
tion of  regional  connective  tissue  which  tends  to  encapsulate  and  then 
restrict  the  progress  of  the  lesion.2 

The  diagnosis  of  blastomycetic  infections  is  best  made  by  a  micro- 
scopical examination  of  the  contents  of  a  papule  or  pustule  teased 
out  in  diluted  NaOH,  and  unstained. 

1  Centralbl.  f.  Bakt.,  1895,  xviii,  521. 

2  Christensen  and  Hektoen,  Jour.  Am.  Med.  Assn.,  1906,  xlvii,  247.     Davis,  Jour.  Inf. 
Dis.,  1911,  viii,  190. 


CHAPTER  XXIX. 

FILTERABLE  VIRUSES,  DISEASES  OF  UNKNOWN 
ETIOLOGY. 

FILTERABLE  VIRUSES.  I  DISEASES  OF  UNKNOWN  ETIOLOGY.  • 

Acute    Anterior    Poliomyelitis.     Epi- j      Measles. 

demic  Poliomyelitis.  Scarlet  Fever. 

Typhus     Fever     (Tabardillo,     Brill's  j      Rabies. 


Disease). 
Yellow  Fever. 
Foot  and  Mouth  Disease. 
Contagious  Pleuropneumoniaof  Cattle. 


Trachoma. 

Smallpox  (Variola)  and  Vaccinia. 

Dengue. 

Rocky  Mountain  Spotted  Fever. 


Mumps. 

FILTERABLE   VIRUSES.1 

THE  viruses  of  certain  diseases  of  plants,  animals  and  of  man  are 
fully  virulent  after  they  have  been  passed,  suspended  in  fluid,  through 
filters  of  unglazed  porcelain  or  diatomaceous  earth  of  definite  degrees 
of  fineness.  These  filters  will  not  permit  the  passage  of  organisms 
as  minute  as  Micrococcus  melitensis,  but  Wherry2  has  shown  that 
the  bacillus  of  guinea-pig  pneumonia,  an  actively  motile  bacillus  0.3 
to  0.5  micron  in  diameter  and  0.7  micron  in  length,  will  also  pass 
through  such  filters  unharmed. 

The  restraining  action  of  filters  of  unglazed  porcelain  and  diato- 
maceous earth  appears  to  depend  rather  upon  the  tortuous  passages 
in  the  walls  of  the  filter  than  upon  the  ultimate  minuteness  of  these 
channels.  This  possibility  is  suggested  by  experiments  using  filters 
of  theoretically  equal  degrees  of  fineness  of  material,  but  of  varying 
thickness;  it  has  been  shown  that  bacteria  may  be  forced  through 
the  thinner  walled  filter,  but  not  through  the  thicker.  Longer  bacteria, 
Bacillus  typhosus  for  example,  will  pass  through  filters,  provided  time 
enough  for  their  development  is  given.  The  supposition  is  that  the 
organisms  grow  around  and  through  tortuous  passages  which  effec- 
tually hold  the  bacteria  in  the  channels  when  pressure  is  applied.  For 
this  reason  filtration  must  not  be  prolonged  much  more  than  an  hour, 
and  too  much  pressure  (or  suction)  must  be  avoided. 

1  See  Wolbach,  Jour.  Med.  Research,  1913,  xxvii,  1,  for  resume  of  literature. 

2  Jour.  Med.  Research,  1902,  viii,  322. 


556 


FILTERABLE   VIRUSES 


The  passage  of  a  virus  through  a  filter  of  the  type  mentioned  does 
not  necessarily  indicate  that  the  virus  is  too  small  to  be  visible  with 
the  highest  powers  of  the  microscope,  although  the  filtrates  of  the 
so-called  "  ultramicroscopic  viruses"  are  clear  and  do  not  contain 
particles  demonstrable  with  the  ultramicroscope.  Filters  used  for 
the  study  of  filterable  viruses  should  be  new,  sterile,  and  tested  for 
permeability  with  suitable  known  bacteria.  A  preliminary  test, 
forcing  air  under  pressure  through  the  submerged  filter,  will  reveal 
"pin  holes."  The  virus  to  be  tested  should  be  forced  through  at  a 


FIG.  91  FIG.  92  FIG.  93  FIG.  94 

FIGS.  91  to  94. — Types  of  unglazed  porcelain  filters.     (Park.) 

temperature  of  about  20°  C.,  and  the  process  should  be  completed 
within  one  and  a  half  hours,  using  as  little  pressure  or  suction  as 
possible.  The  filtrate,  proved  to  be  free  from  visible  particles  (best 
by  adding  a  known  organism  to  the  fluid  to  be  filtered),  should  repro- 
duce the  disease  in  susceptible  animals;  the  virus  should  be  recovered, 
again  filtered,  and  again  reproduce  the  disease.  Some  of  the  filterable 
viruses  will  pass  only  the  coarser  filters,  others  go  through  those  with 
finer  pores. 

Ultramicroscopic  viruses  with  few  exceptions  are  of  unknown 
morphology,  and,  with  the  exception  of  their  resistance  to  desiccation 
and  physical  agents,  but  little  is  known  about  them.  The  viruses  of 


PLATE  VI 


FIG.  1  FIG.  2 

eroorganism  Causing  Epidemic  Poliomyelitis.    (Flexner  and  Noguchi. 

Culture  in  ascitie  fluid-tissue  medium  of 


ACUTE  ANTERIOR  POLIOMYELITIS  557 

pleuropneumonia  of  cattle  and  of  poliomyelitis  have  been  cultivated 
on  artificial  media;  thus  far  the  remainder  have  resisted  attempts  at 
cultivation. 

Acute  Anterior  Poliomyelitis.  Epidemic  Poliomyelitis. — Epidemic 
poliomyelitis  is  an  acute  disease  observed  more  frequently  in  children, 
although  adults  are  by  no  means  immune.  The  onset  is  usually 
abrupt,  although  in  some  cases  the  earliest  symptom  is  fever,  with 
or  without  sore  throat.  The  most  striking  feature  is  a  paralysis  of 
one  or  more  limbs,  which  may  be  the  first  clinical  indication  of  the 
disease.  The  principal  lesion  of  the  earlier  stages  is  a  hyperemia  of 
the  vessels  of  the  cord  together  with  thrombosis,  and  leukocytic  infil- 
tration of  the  perivascular  lymph  spaces,  more  commonly  in  the 
cervical  and  lumbar  regions,  and  in  the  spinal  fluid  as  well.  The  older 
lesions  are  essentially  a  degeneration  of  the  ganglion  cells,  particularly 
of  the  anterior  horn,  and  eventually  their  atrophy.  The  motor  nerves 
appear  to  suffer  most — there  are  few,  if  any,  indications  of  sensory 
disturbance.  The  relation  of  the  disease  to  Landry's  ascending 
paralysis,  if  any,  is  unknown. 

The  etiology  of  acute  anterior  poliomyelitis  was  for  many  years  a 
matter  of  conjecture.  In  1909,  however,  Landsteiner  and  Popper1 
transmitted  the  disease  to  two  monkeys  through  the  injection  of  a 
saline  emulsion  of  the  spinal  cord  from  an  acute  case.  The  animals 
developed  paralysis  of  their  limbs,  and  were  killed  and  studied  bac- 
teriologically  and  pathologically.  The  lesions  were  similar  to  those 
found  in  human  cases;  the  cultures  were  wholly  negative.  An  attempt 
to  introduce  the  disease  in  other  monkeys  by  the  injection  of  material 
from  the  two  successfully  inoculated  animals  proved  futile.  They 
believed  the  virus  belonged  to  the  group  of  filterable  viruses.  Flexner 
and  Lewis2  and  Landsteiner  and  Levaditi3  soon  confirmed  the  filter- 
able nature  of  the  virus,  and  Flexner  and  Lewis  succeeded  in  trans- 
mitting the  virus  through  a  succession  of  monkeys.  The  success  of 
their  transmission  lies  in  the  choice  of  inoculation  site — intracerebral 
inoculations  are  reliable,  but  intraperitoneal  injections  are  usually 
barren  of  results.  Of  great  importance  are  the  observations  of  Flexner 
and  Clark4  and  Osgood  and  Lucas5  that  the  virus  may  survive  in  the 
mucosa  of  the  nasopharynx  of  infected  monkeys  for  several  weeks. 

1  Ztschr.  f.  Immunitatsforsch.,  1909,  ii,  378. 

2  Jour.  Am.  Med.  Assn.,  1909,  liii,  2095. 

3  Compt.  rend.  Soc.  biol.,  1909,  Ixvii,  592. 
4Proc.  Soc.  Exper.  Biol.  and  Med.,  1912,  13,  x,  1. 
5  Jour.  Am.  Med.  Assn.,  1911,  Ivi,  495. 


558  FILTERABLE   VIRUSES 

Landsteiner,  Levaditi  and  Pastia1  have  established  the  presence  of 
the  virus  in  the  tonsils  and  pharyngeal  mucosa  of  an  acute  fatal  case 
of  infantile  paralysis.  Flexner,  Clark  and  Fraser2  have  shown  defi- 
nitely that  the  virus  was  carried  in  the  upper  respiratory  mucous 
membranes  of  healthy  human  adults,  the  parents  of  a  child  suffering 
from  an  acute  attack  of  the  disease.  Kling,  Wernstedt  and  Patterson3 
claim,  on  the  basis  of  experimental  evidence,  that  the  nasal  secretion 
may  also  harbor  the  virus.  Neustaedter  and  Thro4  have  found  that 
the  virus  may  remain  viable  in  dust.  The  transmission  of  the  virus, 
therefore,  would  appear  to  be  largely  through  the  upper  respiratory 
tract.  Flexner  and  Amoss5  have  brought  forth  experimental  evidence 
to  show  that  the  atrium  of  infection  is  the  upper  respiratory  mucous 
membrane,  and  that  the  virus  travels  to  the  meninges  by  way  of  the 
lymphatics;  not,  as  a  rule,  through  the  blood.  Available  evidence 
would  indicate  that  insects  play  no  part,  or  at  best,  a  very  minor 
role  in  the  transmission  of  the  virus.6  The  observations  of  Flexner 
and  Amoss7  and  of  Clark,  Fraser  and  Amoss8  would  indicate  that 
the  amount  of  virus  circulating  in  the  blood  stream  is  usually  very 
small,  thus  suggesting  the  improbability  of  insect  transmission  except 
in  unusual  instances. 

A  very  important  advance  in  the  study  of  the  etiology  of  epidemic 
poliomyelitis  was  that  of  Flexner  and  Noguchi.9  Using  the  technic 
of  Noguchi10  for  the  cultivation  of  Treponemata  (unheated  ascitic 
fluid  and  fragments  of  sterile  rabbit  tissue  under  strictly  anaerobic 
conditions)  they  obtained  minute,  slowly  growing  colonies  composed 
of  "globular  and  globoid"  bodies  occurring  singly,  in  pairs,  masses, 
and  in  short  chains.  The  elements  measure  from  0.15  to  0.3  micron 
in  diameter.  Bizarre  forms  are  prone  to  appear  in  older  cultures. 
The  organisms  stain  feebly  with  the  Giemsa  stain  and  by  Gram's 
method — they  stain  variably  with  the  latter.  The  organism  has  also 
been  demonstrated  in  tissues  by  a  modified  Giemsa  technic.  The 
first  cultivations  upon  artificial  media  are  difficult  to  obtain,  but 
subcultures  grow  more  readily.  No  action  was  observed  on  the 

1  Semaine  Medicale,  1911,  296. 

2  Jour.  Am.  Med.  Assn.,  1913,  Ix,  201. 

3  New  York  Med.  Jour.,  1911,  xciv,  813. 

4Ztschr.  f.  ImmunitatsforscH.,  1911,  xii,  316,  357;    1912,  xiv,  303. 
&  Jour.  Exp.  Med.,  1914,  xx,  249. 

6  Howard  and  Clark,  Jour.  Exp.  Med.,  1912,  xvi,  850.     Sawyer  and  Herms,  Jour.  Am. 
Med.  Assn.,  1913,  Ixi,  461.     Clark,  Fraser,  and  Amoss,  Jour.  Exp.  Med.,  1914,  xix,  223 

7  Jour.  Exp.  Med.,  1914,  xix,  411.  8  Loc.  cit. 
»  Jour.  Am.  Med.  Assn.,  1913,  Ix,  362;  Jour.  Exp.  Med.,  1913,  xviii,  461. 

1°  Jour.  Exp.  Med.,  1911,  xiv,  99;  1912,  xv,  90;   xvi,  199,  211. 


PLATE  VII 


FIG. 


•£/*•: 


FIG.  2 


Survival  and  Virulence  of  Poliomyelitic  Microorganism. 
(Flexner,  Noguehi,  and  Amoss.) 

FIG.  1. — Sediment  showing  the  minute  microorganisms  after  three  days'  growth  in  mixed 
ascitic  fluid  and  bouillon  in  a  flask  employed  for  mass  cultivation.  Giemsa  stain.  X  1000. 

FIG.  2. — Spinal  cord  showing  meningea!  cellular  infiltration  extending  into  the  anterior 
median  fissure.  X  1000. 


PLATE  VIII 


Etiology  of  Epidemic  Poliomyelitis.     (Amoss.) 

FIG.  1. — Globoid  bodies  in  chain  formation  in  brain  tissue  after  six  days'  incubation.  X  1000. 

*  IG.  2. — Globoid  bodies  in  chain  formation  in  brain  tissue  after  eight  days'  incubation.  X  1000. 

FIG.  3. — Globoid  bodies  in  chain  formation  in  brain  tissue  after  ten  days'  incubation.  X  1000 

FIG.  4.— Globoid  bodies  in  mass  formation  in  brain  tissue  after  thirty  days'  incubation.  X  1000. 
FIG.  5. — Globoid  bodies  in  chain  formation  in  heart's  blood.      X  1000. 


TYPHUS  FEVER  559 

ordinary  sugars  and  alcohols.  Growth  appears  to  take  place  in 
litmus  milk  reenforced  with  bits  of  sterile  tissue,  but  no  visible  change 
in  the  medium  can  be  detected.  Cultivations  can  be  readily  made 
from  Berkefeld  filtrates  of  ascitic  fluid  growths,  thus  showing  that 
the  organisms,  or  at  least  some  of  them,  are  filterable.  Cultures  of 
the  organism  were  shown  to  cause  the  typical  disease  with  characteristic 
lesions  in  monkeys. 

Flexner,  Noguchi  and  Amoss1  have  shown  that  cultures  of  the 
organism  may  retain  their  virulence  for  monkeys  at  least  a  year,  and 
Flexner,  Clark  and  Amoss2  have  shown  that  the  virus  retains  its  patho- 
genicity  in  50  per  cent,  glycerin  for  eleven  months;  in  0.5  phenol  for 
five  days,  and  frozen  at  —  2°  to  —  4°  C.  for  at  least  six  weeks.  Amoss3 
has  improved  the  technic  for  cultivating  the  virus  of  epidemic  polio- 
myelitis. 

Pieces  of  brain  from  infected  animals  are  incubated  in  the  kidney- 
ascitic  fluid  of  Noguchi  for  about  two  weeks,  then  crushed  carefully 
and  reincubated  for  three  days  longer.  The  globoid  bodies  appear 
to  multiply  in  the  brain  tissue  and  their  subsequent  recognition  and 
cultivation  is  rendered  more  certain.  Stained  sections  of  such  brain 
tissues  show  increased  numbers  of  organisms. 

Immunity. — One  attack  appears  to  confer  immunity  in  man,  but  the 
evidence  is  not  conclusive.  Flexner  and  Lewis4  have  been  unsuccessful 
in  reinfecting  monkeys  which  have  recovered  from  a  typical  infection 
and  they  lean  toward  the  view  that  one  attack  confers  immunity  in 
these  animals. 

The  characteristic  disease  has  not  been  produced  in  experimental 
animals  other  than  primates. 

Typhus  Fever  (Tabardillo,  Brill's  Disease).— Typhus  fever  is  an 
acute,  febrile  disease  of  man  characterized  by  an  incubation  period 
varying  from  four  to  five  days  to  twelve  days,  an  acutely  developing 
febrile  reaction  which  persists  for  about  two  weeks,  falling  by  crisis, 
or  rapid  lysis,  and  an  extensive  erythematous  eruption,  maculo- 
papular  in  character,  which  appears  usually  within  three  to  four 
days  after  the  onset,  and  persists  for  about  ten  to  fourteen  days. 
The  disease  pathologically  is  to  be  regarded  as  a  hemorrhagic  septi- 
cemia;  the  lesions  postmortem  are  not  distinctive,  and  the  changes 
in  the  organs  are  those  produced  by  an  intense  febrile  reaction.  The 

1  Jour.  Exp.  Med.,  1915,  xxi,  91.  2  Ibid.,  1914,  xix,  207. 

3  Ibid.,  1914,  xix,  212.  -  <  Loc.  cit. 


560  FILTERABLE   VIRUSES 

mortality  varies  greatly;  in  the  eastern  part  of  the  United  States  the 
disease  is  mild  in  character,1  so  mild  in  fact  that  the  malady  was  spoken 
of  as  Brill's  disease  in  honor  of  Brill  who  described  the  clinical  features 
of  it  in  great  detail.  The  mortality  in  Europe,  where  the  disease  is 
very  prevalent  in  certain  areas,  especially  the  more  southern  lands,  is 
usually  high. 

The  first  definite  communication  relating  to  the  mechanism  of 
infection  was  that  of  Nicolle2  and  of  Nicolle,  Comte  and  Conseil.3 
They  succeeded  in  infecting  an  anthropoid  ape  with  the  blood  of  a 
typhus  patient,  and  very  shortly  afterward,  and  independently, 
Anderson  and  Goldberger4  infected  two  monkeys,  a  Macacus  rhesus 
and  a  capuchin,  in  the  same  manner.  These  results  have  been  con- 
firmed by  Ricketts  and  Wilder,5  and  others.  Anderson6  states  that 
guinea-pigs  may  also  be  infected  with  the  blood  of  typhus  patients. 

It  has  been  shown  by  animal  experimentation  that  one  attack 
of  typhus  confers  immunity,  and  this  method  has  been  taken  advantage 
of  to  show  that  typhus,  Brill's  disease  and  tabardillo  mutually 
confer  immunity  on  monkeys;  that  is,  an  animal  recovered  from 
either  of  the  three  clinical  types  is  immune  to  infection  with  the  other 
two. 

The  filterability  of  the  virus  of  typhus  has  been  a  subject  of  discus- 
sion; the  concensus  of  opinion  appears  to  be  that  blood  serum  filtered 
through  stone  filters  has  not  been  definitely  shown  to  be  infective 
for  monkeys,  although  Nicolle,  Anderson  and  Goldberger,  and  Rick- 
etts and  Wilder  have  noticed  that  the  injection  of  filtered  serum  appears 
to  render  monkeys  refractory  to  subsequent  inoculation  with  the 
virus. 

Recently  Plotz7  has  isolated  a  small,  anaerobic,  Gram-positive 
bacillus  from  the  blood  of  a  series  of  cases  of  Brill's  disease  and  of 
typhus  which  when  used  as  an  antigen  caused  fixation  of  comple- 
ment with  the  sera  of  these  cases.  The  bacillus  measures  from  0.2 
to  0.6  micron  in  diameter  and  from  0.9  to  2  microns  in  length.8  It 
is  non-acid-fast,  possesses  no  capsule,  and  exhibits  bipolar  staining. 
In  the  latter  respect  it  suggests  the  organism  seen  but  not  cultivated 

1  Brill,  Am.  Jour.  Med.  Sc.,  April,  1910,  484;  August,  1911,  196. 

2  Compt.  rend.  Acad.  sci.,  1909,  cxlix,  157. 

3  Ibid.,  p.  486. 

4  Public  Health  Rep.,  1909,  1861;   ibid.,  p.  1941;    1910,  177. 

5  Jour.  Am.  Med.  Assn.,  1910,  liv,  463;   ibid.,  1304,  1373. 
«  Public  Health  Rep.,  1915,  xxx,  1303. 

7  Jour.  Am.  Med.  Assn.,  1914,  Ixii,  1556. 

8  Plotz,  Jour.  Am.  Med.  Assn.,  1914,  Ixii,  1556. 


YELLOW  FEVER  561 

by  Ricketts  and  Wilder,1  both  in  the  blood  of  patients  and  in  the 
intestinal  contents  of  lice  which  had  been  permitted  to  bite  these 
patients. 

The  injection  of  cultures  of  the  Plotz  bacillus  into  guinea-pigs 
resulted  after  an  incubation  period  of  from  twenty-four  to  forty- 
eight  hours  in  a  febrile  reaction  which  dropped  by  lysis  after  four  to 
five  days.  This  organism,  Bacillus  typhi-exanthematicus,  as  it  has 
been  named,  must  be  regarded  tentatively  as  the  etiological  factor 
of  typhus  fever. 

Typhus  is  transmitted  by  the  body  louse  Pediculus  vestimenti,  as 
was  shown  by  Nicolle,  Anderson  and  Goldberger,  and  Ricketts  and 
Wilder. 

Yellow  Fever. — Yellow  fever  is  an  acute  fever  of  tropical  and  sub- 
tropical countries,  characterized  by  jaundice,  albuminuria,  and  a 
tendency  to  hemorrhage  from  mucous  membranes;  the  latter  is 
especially  marked  in  the  stomach  and  the  "black  vomit"  which  occurs 
frequently  is  a  regurgitation  of  altered  blood  which  has  collected  in 
the  stomach. 

For  many  years  the  etiology  and  mode  of  transmission  of  yellow 
fever  were  wholly  unknown,  although  many  and  divers  organisms  were 
reported  as  the  inciting  factor.  Finlay,2  as  early  as  1882,  believed 
that  mosquitoes  played  an  important  part  in  the  transmission  of  the 
disease  and  he  actually  attempted  to  infect  non-immunes  by  mos- 
quitoes which  had  previously  bitten  yellow  fever  patients.  His 
experiments  were  wholly  negative,  partly  because  the  extrinsic  cycle 
of  development  in  the  insect  was  unknown.  Carter3  made  the  very 
important  observation  that  a  latent  period  of  about  two  weeks  elapses 
between  primary  and  secondary  cases  of  yellow  fever.  This  discovery 
explained  some  of  Finlay's  negative  results  and  paved  the  way  for  the 
success  of  the  American  Yellow  Fever  Commission.  Finally  Reed, 
Carroll,  Agramonte  and  Lazear,4  a  commission  appointed  from  the 
Medical  Corps  of  the  United  States  Army,  carried  out  a  series  of 
experiments  never  excelled  from  a  scientific  standpoint,  which  showed 
conclusively : 

1.  The  virus  of  yellow  fever  circulates  in  the  blood  stream  of  a 
patient  at  least  three  days  after  the  initial  chill.  An  injection  of  blood 

1  Jour.  Am.  Med.  Assn.,  1910,  liv,  1373. 

2  Ibid.,  1901,  xxxvii,  1387. 

3  Public  Health  Rep.,  1905,  xx,  1350;    New  York  Med.  Rec.,  1906,  Ixix,  683. 

4  Jour.  Exp.  Med.,  1900,  v,  215;    Am.  Public  Health  Assn.,  1900,  xxvi,  37;    Boston 
Med.  and  Surg.  Jour.,  1901,  No.  14;    Jour.  Am.  Med.  Assn.,  1901,  xxxvi,  413. 

36 


562  FILTERABLE  VIRUSES 

from  a  patient  at  this  stage  of  the  disease  will  reproduce  the  disease 
in  a  non-immune. 

2.  The  virus  will  pass  through  a  Berkefeld  filter;  it  belongs,  there- 
fore, to  the  group  of  filterable  viruses.     Berkefeld  filtrates  of  the 
blood  will  establish  the  disease  through  a  series  of  cases,  thus  indi- 
cating that  a  living  virus  is  being  perpetuated. 

3.  The  disease  is  transmitted  ordinarily  by  the  bite  of  a  female 
mosquito  belonging  to  the  genus  Aedes.    The  insect  is  now  known  as 
Aedes  calopus.1 

4.  A  patient  is  infective  for  a  mosquito  only  during  the  first  seventy- 
two  hours  after  the  initial  chill  and  onset  of  the  disease. 

5.  A  latent  period,  during  which  the  insect  is  non-infectious,  must 
elapse  before  the  disease  may  be  transmitted  to  a  non-immune  subject 
through  the  bite  of  the  yellow  fever  mosquito. 

6.  One  attack  appears  to  confer  lasting  immunity,  provided  the 
individual  resides  continuously  in  the  tropics. 

The  two  cardinal  features  of  the  transmission  of  yellow  fever — infec- 
tivity  of  the  patient  during  the  first  three  days  of  the  disease,  and  the 
part  played  in  its  transmission  by  the  mosquito,  Aedes  calopus,  were 
immediately  put  to  the  acid  test  of  practical  sanitation  by  Gorgas,2 
first  in  Havana  and  later  in  Panama,  where  he  organized  and  directed 
the  sanitation  of  these  pestilential  cities  along  lines  which  soon  freed 
them  from  yellow  fever  and  other  diseases  of  endemic  origin  as  well. 

The  importance  of  the  work  of  the  American  Yellow  Fever  Com- 
mission and  of  Gorgas  cannot  be  overestimated;  the  completion  of 
the  Panama  Canal  and  the  liberation  of  the  tropics  from  the  dreaded 
yellow  fever  mark  a  new  era  in  Epidemiology  and  Preventive  Medicine. 

Foot  and  Mouth  Disease.3 — Foot  and  Mouth  disease  is  an  acute, 
highly  infectious  exanthematous  disease  which  attacks  cloven-footed 
animals  chiefly.  The  characteristic  eruptions,  which  are  vesicular 
at  first  and  filled  with  a  clear  fluid,  soon  become  grayish,  and  the 
epidermis  sloughs  off,  leaving  a  raw  reddened  surface.  The  eruption 
usually  appears  at  three  distinct  sites — the  mucous  membrane  of  the 
mouth,  the  teats,  and  interdigital  spaces.  The  incubation  period  is 
from  one  to  six  days,  and  little  or  no  immunity  to  subsequent  attacks 
is  conferred  on  an  animal  by  successful  recovery. 

1  The  original  name  of  the  insect  was  Culex  fasciatus;    it  has  been  changed  succes- 
sively to  Stegomyia  fasciata,  Steogomyia  calopus,  and  finally  to  Aedes  calopus. 

2  See  Jour.  Am.  Med.  Assn.,  1906,  xlvi,  322,  for  brief  summary. 

3  For  an  excellent  discussion  of  various  aspects  of  the  disease,  see  the  Cornell  Vet., 
February,  1915,  Foot  and  Mouth  Disease  Number. 


MEASLES  563 

The  milk  of  infected  cows  contains  the  virus,  and  the  disease  is 
transmissible  to  man,  particularly  young  children,  through  raw  or 
imperfectly  pasteurized  milk,  and  possibly  from  butter  and  cheese 
made  from  infected  milk.  The  disease  is  mild,  as  a  rule,  in  older 
children,  but  it  may  be  severe  or  fatal  for  infants. 

The  virus  belongs  to  the  group  of  filterable  viruses  and,  in  its  purest 
state,  is  found  in  the  contents  of  the  vesicles.  Early  in  the  disease  the 
virus  also  circulates  in  the  blood  stream.  Loffler  and  Frosch,1  who 
discovered  the  filterable  nature  of  the  virus,  found  that  the  vesicular 
fluid,  filtered  through  unglazed  porcelain  filters,  retained  its  infectious- 
ness  for  some  time,  provided  the  fluid  be  kept  cool  and  in  the  dark. 

Contagious  Pleuropneumonia  of  Cattle. — This  disease  was  the  first 
to  be  described  in  which  the  virus  passes  through  unglazed  porcelain 
filters,  although  the  filtration  of  the  virus  was  not  attempted  at  thaf 
time.  Xocard  and  Roux2  examined  the  exudate  from  the  lungs  of 
diseased  cattle  microscopically  with  negative  results.  They  suspended 
it  in  broth,  enclosed  in  collodion  capsules,  in  the  peritoneal  cavities 
of  guinea-pigs.  After  two  to  four  weeks  the  medium  became  turbid, 
while  controls  remained  clear.  Examination  of  the  fluid  under  a 
magnification  of  2000  diameters  revealed  very  minute,  highly  refrac- 
tile  spots  which  exhibited  Brownian  movement.  They  claim  to  have 
cultivated  the  virus  in  a  peptone-serum  medium  and  to  have  obtained 
minute  colonies  (0.5  mm.  diameter)  on  peptone-serum  agar.  Later 
the  virus  was  shown  to  pass  through  Berkefeld  filters  and  the  coarser 
grades  of  porcelain  filters,  but  not  the  finer  grades. 

The  disease  is  confined  to  cattle;  man  is  immune  so  far  as  is  known. 

DISEASES   OF   UNKNOWN   ETIOLOGY. 

Measles. — The  etiology  of  measles  is  unknown,  but  Hektoen3 
produced  the  disease  in  two  susceptible  individuals  by  injecting 
blood  from  a  patient  exhibiting  typical  symptoms.  The  blood  was 
removed  about  thirty  hours  after  the  appearance  of  the  eruption,  and 
the  disease  induced  was  clinically  perfectly  typical.  Anderson  and 
Goldberger4  report  a  successful  inoculation  of  several  monkeys  with 
blood  from  human  cases;  four  out  of  a  total  of  nine  animals  developed 
a  febrile  reaction  and  a  limited  eruption.  The  virus  was  carried 
through  three  monkey  generations  in  one  experiment.  Growth  was 

1  Central bl.  f.  Bakt.,  I  Abt.,  1898,  xxiii,  371.  2  Ann.  Inst.  Past.,  1898,  xii,  240. 

3  Jour.  Inf.  Dis.,  1905,  ii,  238.  <  Public  Health  Rep.,  1911,  xxvi,  No.  24. 


564  DISEASES  OF   UNKNOWN  ETIOLOGY 

not  obtained  in  artificial  media  heavily  inoculated  with  blood  from 
patients,  shown  by  experiment  to  contain  the  virus. 

Buccal  and  nasal  secretions  contain  virus  of  measles  which  passes 
a  Berkefeld  filter.1 

Scarlet  Fever. — The  etiology  of  scarlet  fever  is  unknown.  The 
very  common  occurrence  of  streptococci  in  this  disease  has  led  many 
observers  to  attribute  to  the  streptococcus  an  etiological  relationship. 
No  satisfactory  evidence  in  support  of  the  view  that  any  type  of 
streptococcus  is  the  causative  agent  has  been  brought  forward. 

Dohle2  described  small  oval,  round  and  rod-shaped  bodies  measur- 
ing about  1  micron  in  diameter,  lying  within  the  cytoplasm  of  poly- 
morphonuclear  leukocytes  in  a  series  of  cases  of  scarlet  fever.  It  was 
assumed  at  first  that  these  inclusion  bodies  were  fragments  of  a 
spirochete  (the  hypothetical  inciting  agent  of  scarlet  fever)  which  had 
been  phagocytized  and  disintegrated  by  the  polymorphonuclear 
leukocytes.  This  view  is  now  discredited.  Numerous  investigations, 
especially  that  of  Hill,3  indicated  that  the  Dohle  bodies  are  fragments 
of  the  nucleus  of  the  leukocyte,  presumably  a  reaction  to  injury  by 
bacterial  toxins.  They  are  present,  however,  in  a  majority  of  cases  of 
scarlet  fever  up  to  the  tenth  day  and  especially  numerous  during 
the  first  four  days  of  the  clinical  disease,  as  the  following  table  by 
Hill  shows.  The  Poppenheim  stain  (two  parts  of  a  saturated  aqueous 
solution  of  pyrosin  and  four  parts  of  a  saturated  aqueous  solution  of 
methyl  green)  is  especially  recommended  for  the  demonstration  of  the 
inclusion  bodies  of  Dohle.  The  nuclei  of  the  cell  are  colored  greenish 
blue,  the  Dohle  bodies  bright  red. 


Scarlet  fever  

Positive. 
.      .      .      .     434 

Negative. 
295 

Total. 

72 

Erysipelas             .... 

.      .      .      .        5 

0 

5 

Pneumonia      

.      .      .      .        4 

1 

5 

Syphilis     
Empyemia      
Secondary  anemia     . 
Serum  rash     
Normal 

.      .      .      .        0 
.      .      .      .        0 
.      .      .      .        0 
.      .      .      .        0 
0 

2 
1 

1 
1 
13 

2 
1 
1 
1 
13 

Hill  concludes  that  the  Dohle  inclusion  bodies  are  present  in  a 
majority  of  cases  of  scarlet  fever  up  to  the  tenth  day,  but  they  are  not 

1  Goldberger  and  Anderson,  Jour.  Am.  Med.  Assn.,  1911,  Ivii,  476,  971. 

2  Centralbl.  f.  Bakt.,  Orig.,  1911,  Ixi,  63. 

3L.  W.  Hill,  Boston  Med.  and  Surg.  Jour.,  1914,  clxx,  792;  excellent  summary  of 
literature. 

4  25  cases  examined  before  tenth  day;  18  after  tenth  day;  latest  case  forty-fifth  day. 

5  All  except  6  cases  after  tenth  day ;   remaining  6  cases  had  normal  temperature  and 
very  slight  rash. 


RABIES  565 

specific  for  the  disease;  they  are  found  in  other  infections,  especially 
erysipelas,  sepsis,  pneumonia  and  tonsillitis.  They  are  more  likely 
to  be  found  in  disease  with  which  the  streptococcus  is  associated. 
Diagnostically  they  possess  some  value.  If  they  are  not  found  in  a 
doubtful  case  which  has  a  rash  and  a  marked  fever,  the  case  is  prob- 
ably not  one  of  scarlet  fever.1 

Rabies. — Rabies  is  a  disease  primarily  observed  among  the  carnivora 
— dogs,  wolves  and  cats — but  it  is  transmissible  to  horses  and  to  man. 
Laboratory  animals  are  readily  infected  with  the  virus.  The  saliva 
of  rabid  animals  is  infectious  and  the  natural  mode  of  inoculation  is 
through  bites  of  infected  animals.  The  disease  is  also  readily  trans- 
missible in  an  experimental  way  through  the  injection  of  emulsions 
of  the  cord  or  brain  of  rabid  animals  directly  into  the  central  nervous 
system  of  other  animals.  The  infectious  nature  of  rabies  was  first 
clearly  shown  by  Pasteur,  Chamberland  and  Roux.2 

The  incubation  period  for  "street  rabies"  is,  on  the  average,  from 
one  to  two  months,  but  it  may  be  considerably  longer.  The  incidence 
of  the  disease  among  those  bitten  by  rabid  dogs  depends  largely  upon 
the  location  of  the  bite — if  upon  the  body  protected  with  several 
layers  of  clothing,  infection  may  fail  to  develop;  the  virus  is  held 
back  by  the  clothing  and  fails  to  enter  the  wound.  In  general  the 
inoculation  period  is  shortest  when  the  hands  or  face  are  attacked, 
because  the  virus  acts  upon  the  central  nervous  system  and  reaches 
it  through  the  peripheral  nerves. 

The  disease  in  man  is  practically  always  acute  and  death  usually 
terminates  the  infection  within  three  to  six  days  after  the  onset  of 
the  symptoms.  The  initial  symptoms  are  premonitory  and  consist 
typically  of  slight  irritation  at  the  site  of  inoculation,  together  with 
psychic  depression.  The  characteristic  symptoms  are  paralysis  of 
the  muscles  of  deglutition — which  leads  to  extreme  difficulty  in 
swallowing — hyperesthesia,  extreme  restlessness  and  irritability,  and 
violent  reflex  spasms.  Even  so  slight  an  effort  as  that  required  to 
swallow  water  frequently  causes  such  violent  paroxysms  that  the 
mere  sight  of  water  is  distressing — hence  the  name  hydrophobia — the 
dread  of  water.  It  is  important  to  remember  that  the  hydrophobic 
phenomena  are  much  less  commonly  seen  in  rabid  dogs  than  in  man; 
indeed  rabid  dogs  frequently  swim  across  streams  that  they  happen 
to  encounter.  The  final  stage  of  rabies  is  a  progressive  paralysis, 

1  Hill,  loc.  cit. 

2  Compt.  rend.  Acad.  Sc.,  1881.  xcii,  159. 


566  DISEASES  OF  UNKNOWN  ETIOLOGY 

which  usually  first  becomes  manifest  in  the  limbs  and  arms ;  it  ascends 
gradually  and  death  occurs  when  the  higher  centres  are  reached. 

The  disease  occurs  in  every  country  except  England,  and  possibly 
Australia.  The  elimination  of  rabies  from  England  dates  from  the 
law  of  1889  which  required  all  dogs  to  be  muzzled  and  all  imported 
animals  to  be  quarantined  for  several  months.  The  law  was  allowed 
to  lapse  for  a  time,  the  disease  reappeared,  but  a  new  and  rigid  enforce- 
ment of  the  muzzling  and  quarantine  laws  has  completely  eliminated 
rabies  from  the  British  Isles.  No  cases  have  been  reported  since  1903. 

The  first  definite  lesions  characteristic  of  rabies  were  described 
by  Negri,1  who  found  characteristic  cell  inclusion  bodies  in  the  ganglion 
cells,  in  the  cells  of  Purkinje,  and  other  large  nerve  cells.  These  minute 
granular  pleiomorphic  bodies  are  now  recognized  as  specific,  or  nearly 
so,  for  hydrophobia,  but  there  is  discussion  of  their  nature.  Williams2 
regards  them  as  protozoa  and  conferred  upon  them  the  name  Neuror- 
rhyctes  hydrophobise ;  in  collaboration  with  Lowden3  she  has  made  a 
careful  study  of  the  occurrence  of  Negri  bodies  and  considers  them  the 
true  etiological  agent  of  rabies.  Remlinger,4  Poor  and  Steinhardt,5 
and  others  have  found  that  the  virus  is  filterable,  and  Noguchi6  has 
cultivated  an  organism  from  "street"  virus  and  from  the  central 
nervous  system  of  animals  infected  with  "street"  virus,  "fixed" 
virus  and  with  "passage"  virus,  which  resemble  Negri  bodies  observed 
in  lesions  in  many  particulars.  The  smallest  of  these  bodies  are  just 
visible  with  the  highest  magnifications  obtainable;  larger  nucleated 
or  oval  bodies  occasionally  appear  in  older  cultures.  Inoculation  of 
dogs,  rabbits  and  guinea-pigs  with  cultures  containing  the  granular 
pleiomorphic  or  nucleated  bodies  was  followed  by  typical  symptoms 
of  rabies.  The  relation  of  the  organisms  grown  by  Noguchi  to  Negri 
bodies  is  not  definitely  determined  as  yet,  but  the  organism  has  been 
kept  alive  for  over  three  months  in  artificial  cultures  and  found  to 
be  virulent  after  the  twenty-first  transfer  in  artificial  media.  This 
would  suggest  strongly  that  Noguchi's  organism  was  the  etiological 
agent  of  rabies.  The  possibility  that  a  filterable  virus  was  growing 
in  these  cultures  cannot  be  overlooked,  as  Noguchi  has  pointed  out, 
but  there  is  no  evidence  that  such  is  the  case. 

The  most  important  rapid  laboratory  method  for  the  diagnosis  of 

1  Ztschr.  f.  Hyg.,  1903,  xliii,  507;    1909,  Ixiii,  421. 

2  Proc.  New  York  Path.  Soc.,  1906,  vi,  77. 

3  Williams  and  Lowden,  Jour.  Inf.  Dis.,  1906,  iii,  452. 
« Ann.  Inst.  Past.,  1903,  xvii,  834:    1904,  xviii,  150. 

5  Jour.  Inf.  Dis.,  1913,  xii,  202.  6  Jour.  Exp.  Med.,  1913,  xviii,  314. 


RABIES  567 

rabies  is  a  demonstration  of  Negri  bodies.  If  they  are  found  the 
diagnosis  is  complete.  Failure  to  find  them  does  not  necessarily 
exclude  a  diagnosis  of  rabies,  and  an  emulsion  prepared  from  the 
central  nervous  system,  using  the  gray  substance  as  far  as  possible, 
is  injected  subdurally  into  an  experimental  animal  for  a  final  diag- 
nosis. The  method  of  animal  inoculation,  while  slower  than  the 
microscopic  examination  of  the  brain,  is  the  final  test  in  doubtful 
cases.  Of  course,  treatment  should  not  await  the  results  of  animal 
inoculation  if  there  is  suspicion  that  a  patient  has  been  bitten  by  a 
rabid  dog,  especially  if  the  hands,  face  or  other  unprotected  surface 
be  the  site  of  the  wounds. 

Staining  Negri  Bodies. — Williams  and  Lowden1  have  developed  a 
technic  for  the  rapid  demonstration  of  Negri  bodies,  which  is  widely 
followed  at  the  present  time.  A  small  piece  of  the  gray  substance 
from  the  region  of  the  hippocampus  major  and  from  the  cerebellum 
of  the  animal  is  placed  upon  a  clean  glass  slide  and  covered  with  a 
clean  coverglass.  Pressure  is  applied  to  the  latter  until  the  tissue  is 
flattened  and  spread  uniformly.  The  pressure  is  now  shifted  to  one 
edge  of  the  coverglass  and  the  flattened  tissue  is  forced  along  the 
slide,  leaving  a  thin  film  as  it  passes.  Fixation  with  neutral  absolute 
methyl  alcohol  (Merck  reagent)  containing  about  0.1  per  cent,  picric 
acid  (about  ten  minutes  are  required)  is  followed  by  removal  of  the 
fixing  agent  with  filter  paper. 

A  small  amount  of  a  freshly  prepared  staining  mixture,  made  in 
the  proportions  of  30  c.c.  of  distilled  water,  10  c.c.  of  a  saturated  alco- 
holic solution  of  methylene  blue  and  0.5  c.c.  of  a  saturated  alcoholic 
solution  of  basic  fuchsin  is  poured  over  the  slide,  warmed  till  steam 
arises,  then  poured  off.  The  excess  stain  is  removed  in  running  water 
and  the  preparation  is  carefully  dried  with  filter  paper.  The  prepara- 
tion is  examined  with  an  oil  immersion  lens. 

Negri  bodies,  which  vary  in  size  from  about  1  micron  to  25  microns 
in  diameter,  are  stained  magenta  with  blue  granules  by  this  process; 
the  cytoplasm  of  the  nerve  cells  is  pale  blue;  the  nuclei  of  the  nerve 
cells  are  colored  a  darker  blue. 

The  Pasteur  Treatment  for  Rabies. — Pasteur2  made  the  very  important 
observation  that  the  virus  of  rabies  as  it  exists  in  rabid  dogs  (street 
virus)  could  be  so  attenuated  by  repeated  passages  through  rabbits 
that  it  lost  much  of  its  original  virulence  for  the  dog.  This  change  in 

1  Loc.  cit.  t  2  Loc.  cit. 


568  DISEASES  OF   UNKNOWN  ETIOLOGY 

virulence  was  fully  established  when  passage  of  the  virus  from  rabbit 
to  rabbit  caused  each  successive  animal  to  sicken  in  about  six  or  seven 
days,  and  to  die  regularly  on  the  ninth  day.  No  further  increase  in 
pathogenicity  for  the  rabbit  could  be  induced,  and  the  virus  at  this 
level  of  virulence  was  called  "virus  fixe"  by  Pasteur.  The  spinal  cord 
of  such  a  rabbit,  dried  for  two  weeks  over  caustic  soda  at  room  tem- 
perature, lost  its  virulence  for  rabbits,  although  cords  dried  for  a  week 
or  ten  days  killed  the  animal  when  injected  subdurally;  the  period 
of  incubation  was,  of  course,  increased  when  the  partly  dried  cords 
were  used. 

The  original  Pasteur  treatment  consisted  in  grinding  a  piece  of 
dried  cord  half  a  centimeter  in  length  in  5  c.c.  of  sterile  salt  solution, 
and  injecting  the  emulsion  subcutaneously,  preferably  on  the  abdo- 
men of  the  patient.  Daily  injections,  using  fresher  and  fresher  cords 
were  used,  until  finally  a  cord  from  a  rabbit  dead  but  twenty-four 
hours  furnished  the  material  for  inoculation.  The  entire  treatment 
required  about  three  weeks,  at  the  end  of  which  time  a  very  decided 
degree  of  immunity  was  induced.  The  incubation  period  of  the 
naturally  acquired  disease  is  usually  not  less  than  six  weeks;  the 
advantage  of  instituting  treatment  at  the  earliest  possible  moment  is 
obvious. 

The  mortality  from  rabies  among  those  treated  by  the  Pasteur 
method  of  immunization  is  less  than  0.5  per  cent.;  the  average  mor- 
tality of  untreated  cases  is  about  16  per  cent. 

Modifications  in  the  original  Pasteur  treatment,  principally  along 
the  lines  of  injecting  more  virulent  material,  have  been  made  from 
time  to  time,  and  the  tendency  at  present  is  to  administer  a  shorter 
treatment  to  mild  cases  (judged  according  to  the  location  of  the  bite 
and  the  extent  of  local  injury)  on  the  one  hand,  and  to  administer 
a  much  more  intense  treatment  in  the  severe  cases.  The  present 
routine  followed  in  the  Pasteur  Institute  of  Paris  is  shown  in  the 
accompanying  table1  (see  page  569). 

Statistics  indicate  that  a  considerable  degree  of  immunity  is  devel- 
oped by  the  end  of  the  second  week  of  the  treatment.  The  duration 
of  the  immunity  has  not  been  definitely  established,  but  it  appears 
to  last  for  several  years.  Exposure  to  extreme  cold  and  excesses  of 
various  kinds,  especially  alcoholism,  are  said  to  be  dangerous  imme- 
diately after  the  treatment  is  completed;  they  may  reduce  the 

1  Kraus  and  Levaditi,  Handbuch  der  Technik  und  Methodik  der  Immunitatsforschung, 
1908,  i,  713. 


PLATE   IX 


Negri  Bodies. 
Redrawn  from  Kolle  and  Hetsch.     (Lentz  stain.) 


TRACHOMA 


569 


acquired  resistance  to  the  virus  to  such  a  degree  that  the  patient  will 
succumb  to  a  latent  infection. 

The  dangers  attending  the  treatment  are  slight;  in  a  moderate 
number  of  cases  the  sites  of  earlier  injections  may  become  inflamed 
after  the  treatment  has  been  continued  for  ten  days  or  two  weeks, 
but  this  reaction  is  regarded  as  a  modified  Arthus  phenomenon  depend- 
ing upon  local  sensitization.  By  far  the  most  serious  complication 


PASTEUR   INSTITUTE   IMMUNIZATION   FOR   RABIES. 
(KRAUS  AND  LEVADITI.) 


Days. 

Mild  cases. 

Moderate  cases. 

Severe  cases. 

Dried  cord.1 

Amount 

Dried  cord.1 

Amount 

Dried  cord.1 

Amount 

A.M.                           P.M. 

1 

14  +  13  day 

3  c.c. 

14  +  13  day 

3  c.c. 

14  +  13  day   12  +  11  day 

3  c.c. 

2 

12  +  11  day 

3  c.c. 

12  +  11  day 

3  c.c. 

10+9  day     8+7  day 

3  c.c. 

3 

10+9  day 

3  c.c. 

10+9  day 

3  c.c. 

6  day                6  day 

2  c.c. 

4 

8+7  day 

3  c.c. 

8+7  day 

3  c.c. 

5  day 

2  c.c. 

5 

6+6  day 

2  c.c. 

6+6  day 

2  c.c. 

5  day 

2  c.c. 

6     j              5  day 

1  c.c. 

5  day 

2  c.c. 

4  day 

2  c.c. 

7 

5  day 

1  c.c. 

5  day 

2  c.c. 

3  day 

1  c.c. 

8 

4  day 

1  c.c. 

4  day 

2  c.c. 

4  day 

2  c.c. 

9 

3  day 

1  c.c. 

3  day 

1  c.c. 

3  day 

1  c.c. 

10 

5  day 

2  c.c. 

5  day 

2  c.c. 

5  day 

2  c.c. 

11 

5  day 

2  c.c. 

5  day 

2  c.c. 

5  day 

2  c.c. 

12 

4  day 

2  c.c. 

4  day 

2  c.c. 

4  day 

2  c.c. 

13 

4  day 

2  c.c. 

4  day 

2  c.c. 

4  day 

2  c.c. 

14 

3  day 

2  c.c. 

3  day 

2  c.c. 

3  day 

2  c.c. 

15 

3  day 

2  c.c. 

3  day 

2  c.c. 

3  day 

2  c.c. 

16 

5  day 

2  c.c. 

5  day 

2  c.c. 

17 

4  day 

2  c.c. 

4  day 

2  c.c. 

18 

.... 

3  day 

2  c.c. 

3  day 

2  c.c. 

19 

.... 

5  day 

2  c.c. 

20 

4  day 

2  c.c. 

21 

3  day 

2  c.c. 

Injections  daily  for  two  to  three  weeks. 

of  the  treatment  is  a  paralysis  which,  in  rare  instances,  appears  during 
the  progress  of  the  treatment,  or  shortly  afterward.  This  usually 
results  fatally.  The  cause  of  this  paralysis  is  not  definitely  known, 
but  it  is  assumed  that  it  is  a  modified  form  of  the  disease. 

Trachoma. — The  etiology  of  trachoma — contagious  granular  con- 
junctivitis characterized  by  the  formation  of  small  granular  elevations 
of  the  eyelids  that  atrophy  and  lead  to  scar  formation — is  not  defi- 
nitely settled. 


1  One  centimeter  of  cord  of  the  age  indicated,  ground  in  5  c.c.  of  sterile  salt  solution, 
and  injected  as  per  schedule. 


570  DISEASES  OF   UNKNOWN  ETIOLOGY 

Halberstadter  and  Prowazek1  have  described  endocellular  bodies 
lying  within  the  conjunctival  epithelium  and  usually  near  the  cell 
nuclei,  which  are  minute  oval  or  round  granules  frequently  occurring 
in  pairs,  of  somewhat  variable  size,  but  smaller  than  ordinary  cocci. 
They  are  typically  enclosed  in  a  somewhat  indefinitely  defined  homo- 
geneous matrix  which  is  regarded  as  a  reaction  product.  The  earlier 
lesions  contain  moderate-sized  oval  or  round  bodies  which  stain  a 
faint  bluish  color  with  Giemsa's  stain;  later  very  minute  oval  or 
spherical  bodies  appear,  which  color  reddish  with  the  same  stain. 
These  observations  were  soon  confirmed.  Somewhat  later  the  same 
investigators  described  inclusions  in  the  conjunctival  epithelium  of 
uncomplicated  cases  of  blennorrhea  neonatorum  which  were  prac- 
tically identical  histologically  with  those  described.  This  observation 
naturally  led  to  new  investigation  of  the  subject. 

Berterelli  and  Cecchetto2  claimed  to  have  reproduced  trachoma  in 
a  Macacus  monkey  with  a  filtrate  (Berkefeld)  prepared  from  a  human 
case.  Nicolle,  Guenod  and  Blaisot3  were  unable  to  infect  monkeys, 
but  stated  that  anthropoid  apes  were  susceptible  to  the  trachoma  virus. 

Herzog4  believed  that  the  "trachoma  bodies"  were  involution  forms 
of  the  gonococcus  which,  under  certain  unknown  conditions,  develops 
into  very  small  forms  that  are  indistinguishable  from  the  trachoma 
bodies  when  they  are  within  the  epithelial  cells.  Herzog  claims  to 
have  developed  these  very  minute  forms  (microgonococci)  in  artifi- 
cial media  through  a  series  of  rapid  transplantations,' and  he  states 
that  this  minute  state  in  the  development  of  the  organism  is  the  one 
which  leads  to  trachoma.  Williams5  has  studied  trachoma  extensively 
and  believes  that  the  cellular  inclusions  characteristic  of  trachoma  are 
degenerated  hemoglobinophilic  bacilli.  Noguchi  and  Cohen6  have 
cultivated  an  organism  from  cases  of  conjunctivitis  in  which  the 
inclusion  bodies  were  present,  and  from  an  older  case  in  which  no 
inclusion  bodies  were  found,  which  repeats  in  culture  many  of  the 
important  morphological  appearances  of  the  trachoma  bodies.  It  is 
certainly  neither  a  gonococcus  nor  a  member  of  the  group  of  hemo- 
globinophilic bacteria,  but  its  identity  with  the  trachoma  bodies  is 

1  Deutsch.  mod.  Wchnschr.,  1907,  xxxiii,  1285;  Arb.  a.  d.  Kais.  Gesundheitsamte,  1907, 
xx vi,  44. 

2  Centralbl.  f.  Bakt.,  Orig.,  1908,  xlvii,  432. 

3  Compt.  rend.  Aead.  sc.,  1911,  clii,  1504. 

4  Centralbl.  f.  Bakt.,  Ref.,  1910,  xlviii,  276;  Arch.  f.  Ophth.,  1910,  Ixxiv,  520;  Ueber 
die  Natur  und  Herkunft  d.  Trachomaerregers,  Berlin  and  Wien,  1910. 

6  Arch.  Ophth.,  1913,  xlii,  506;  Jour.  Inf.  Dis.,  1914,  xiv,  261. 
6  Jour.  Exp.  Med.,  1913,  xviii,  572;  1915,  xxii,  304. 


SMALLPOX  AND  VACCINIA  571 

not  yet  determined  by  its  discoverers.  Noguchi  and  Cohen  have 
made  the  important  observation  that  the  conjunctive  of  certain 
monkeys  are  susceptible  to  infection  with  material  containing  the 
von  Prowazek  inclusion  bodies,  but  not  to  the  hemoglobinophilic 
bacilli  isolated  from  cases  of  epidemic  conjunctivitis;  on  the  other 
hand,  pure  cultures  of  hemoglobinophilic  bacilli  cause  an  acute 
inflammation  in  the  testes  of  rabbits;  at  certain  stages  of  the  infection 
numerous  clumps  of  the  organisms  occur,  which  stimulate  the  von 
Prowazek  cell  inclusions.  Injection  of  conjunctival  scrapings  con- 
taining the  cell  inclusion  bodies  alone  is  without  effect  in  the  rabbit. 

These  observations  have  led  Noguchi  and  Cohen  to  conclude  that 
a  group  of  cases  exists  in  which  epithelial  cell  inclusions  alone  may 
be  demonstrated  in  smears;  pneumococci  and  hemoglobinophilic 
organisms  are  absent.  The  conjunctiva  may  become  infected  both 
with  the  inclusion  bodies  and  hemoglobinophilic  organisms.  The 
susceptibility  of  the  conjunctiva  of  certain  monkeys  to  infection  with 
the  hemoglobinophilic  bacilli  would  appear  to  be  an  important  method 
for  diagnosis  of  the  von  Prowazek  inclusion  bodies. 

Smallpox  (Variola)  and  Vaccinia. — Smallpox  (variola)  and  vaccinia, 
now  generally  regarded  as  an  infection  produced  by  the  virus  of  small- 
pox modified  by  successive  passages  through  the  cow,  are  of  unknown 
etiology.  Guarnieri1  has  observed  and  described  cell  inclusions  in 
the  epithelia  of  both  smallpox  and  vaccinia  lesions  and  in  experi- 
mental lesions  in  the  cornea  of  rabbits  as  well,  which  he  regards  as 
protozoa,  and  to  which  he  gave  the  name  Cytoryctes  variola?.  Coun- 
cilman, Magrath  and  Brinkerhoff2  have  studied  these  vaccine  bodies 
in  detail  and  incline  to  the  view  that  they  are  parasites  specific  for 
the  disease.  Calkins3  has  construed  the  various  forms  of  the  cell 
inclusions  to  be  distinct  stages  in  the  life  history  of  a  protozoal  parasite. 
The  protozoal  nature  of  the  "vaccine  bodies"  is  not  universally  con- 
ceded, and  the  conservative  statement  of  Ewing4  that  they  may  be 
regarded  as  degenerative  phenomena  characteristic  for  the  disease  is 
widely  accepted  at  the  present  time. 

The  close  relationship  between  smallpox  and  vaccinia  (cowpox) 
has  been  recognized  since  Jenner's5  classical  researches  published  in 

1  Centralbl.  f.  Bakt.,  1894,  xvi,  299.  2  Jour.  Med.  Research,  1904,  xi,  12. 

3  Ibid.,  p.  136.  4  Jour.  Med.  Research,  xiii,  233. 

6  An  Inquiry  Into  the  Causes  and  Effects  of  the  Variolse  Vaccinise,  a  disease  discovered 
in  some  of  the  Western  Counties  of  England,  particularly  Gloucestershire,  and  Known 
by  the  Name  of  the  Cow  Pox,  London,  Sampson  Low,  1789.  (See  Epoch-making 
Contributions  to  Medicine,  Surgery,  and  Allied  Sciences,  Carmac,  Saunders  and  Co.) 


572  DISEASES  OF   UNKNOWN  ETIOLOGY 

1789;  he  showed  experimentally  that  a  successful  inoculation  of  man 
with  cowpox  virus  protected  the  individual  against  infection  with 
the  virus  of  smallpox. 

The  change  which  the  smallpox  virus  undergoes  during  passage 
through  calves  is  not  definitely  known,  but  Councilman,  Magrath, 
Brinkeroff  and  others  are  of  the  opinion  that  the  smallpox  virus 
is  somewhat  widely  distributed  in  the  viscera  and  different  organs 
of  the  body  (in  man);  passage  of  the  virus  through  calves  so  modifies 
its  activities  that  it  localizes  rather  specifically  in  pavement  epithelium. 
The  relatively  insignificant  local  lesions  of  vaccinia  in  contrast  to  the 
general  distribution  of  the  eruption  and  lesions  of  smallpox  are  in 
harmony  with  this  view. 


FIG.  95. — Guarnieri  cell  inclusion  bodies. 

Jenner's  remarkable  studies  upon  the  immunity  to  smallpox  that 
follows  vaccination  with  cowpox  virus  have  been  amply  confirmed 
by  the  observations  of  Brinkerhoff  and  Tyzzer,1  who  showed  that 
vaccination  of  monkeys  protects  them  from  subsequent  infection 
with  the  smallpox  virus. 

Originally  vaccine  virus  was  perpetuated  by  arm  to  arm  inocula- 
tion, but  the  danger  of  transmitting  syphilis  or  other  disease  as  well 
as  the  uncertainty  of  the  method  have  led  to  the  use  of  calves  as  a  source 
of  vaccine  virus. 

The  source  of  the  virus  is  threefold:2 

1.  Virus  descended  from  spontaneous  cowpox  and  continued  through 
an  indefinite  series  of  animals — the  true  animal  vaccine. 

1  Jour.  Med.  Research,  1905,  xiv,  209. 

2  Theobald  Smith,  Med.  Soc.  Proc.,  June  10,  1903. 


SMALLPOX  AND   VACCINIA  573 

2.  Virus  obtained  from  animals  which  have  been  inoculated  with 
lymph  from  human  vaccine  pustules,  either  directly  or  indirectly, 
through  a  series  of  calves — this  is  known  as  retrovaccine. 

3.  Vaccine  obtained  by  passing  smallpox  virus  through  the  cow— 
the  so-called  variola  vaccine. 

Preparation  of  Vaccine  Virus. — Healthy  female  calves  about  three 
months  of  age  are  selected.  After  thorough  cleansing  the  animal  is 
fastened  upon  an  operating  table  of  special  design  and  the  abdomen 
and  inner  aspect  of  the  thighs  are  shaved.  If  disinfectants  have  been 
used  they  are  removed  with  sterile  water.  Shallow  parallel  incisions 
about  half  an  inch  apart  and  just  deep  enough  to  become  slightly 
reddened  are  made,  and  the  vaccine  is  thoroughly  rubbed  into  the 
scarified  area.  The  quarters  in  which  inoculated  calves  are  kept  are 
scrupulously  clean;  the  animals  are  preferably  fed  an  exclusive  milk 
diet.  Dust  is  reduced  to  a  minimum  and  excreta  are  promptly  removed 
by  flushing  with  a  stream  of  water. 

Four  to  six  days  after  inoculation,  depending  upon  the  rate  of 
development  of  the  vaccine  vesicles,  the  calf  is  again  placed  upon 
the  table,  the  vaccinated  area  washed  with  sterile  water  and  then 
rubbed  gently  with  sterile  absorbent  cotton;  any  crusts  or  scabs  are 
removed.  The  slightly  elevated  confluent  eruption  is  curetted  away 
and  appears  as  a  pulpy  mass,  which  is  thoroughly  ground  in  a  mill 
of  special  design  with  three  or  four  times  its  volume  of  60  per  cent. 
glycerin.1  The  ground  and  comminuted  glycerized  virus  thus  pre- 
pared contains  variable  numbers  of  bacteria;2  as  many  as  700,000 
per  c.c.  have  been  found.3  Of  the  more  common  microorganisms, 
various  molds,  yeasts  and  members  of  the  coccal  group  are  usually 
present.  Very  rarely  cases  of  tetanus  have  been  reported  following 
vaccination.4  The  extreme  rarity  of  these  cases  and  the  possibility 
of  infection  from  uncleanly  conditions  after  the  vaccination  was 
made  make  it  doubtful  that  vaccine  may  be  a  vehicle  for  the  trans- 
mission of  tetanus.5 

The  addition  of  the  glycerin  to  the  pulp  obtained  from  vaccinated 
calves  plays  an  important  part  in  reducing  the  number  of  bacteria 

1  Carbolic  acid  (1  per  cent.)  is  frequently  added  to  the  glycerin  before  mixing  it   with 
the  pulp;    experience  indicates  that  the  carbolized  vaccine  virus  loses  its  potency  more 
rapidly  than  when  glycerin  alone  is  used. 

2  See  Rosenau,  Am.  Med.,  1902,  iii,  637,  for  Bacteriology. 

3  Theobald  Smith,  loc.  cit. 

4  Wilson,  Jour.  Am.  Med.  Assn.,  1902,  xxxviii,  1147,  1222.     McFarland,  Jour.  Med. 
Research,  1902,  vii,  474. 

5  See  Francis,  Bull.  No.  95,  U.  S.  P.  H.  and  Marine  Hosp.  Service,  1914,  for  results  of 
implanting  tetanus  spores  directly  into  vaccine. 


574  DISEASES  OF   UNKNOWN  ETIOLOGY 

which  are  invariably  present  in  "green  vaccine" — it  does  not  seriously 
impair  the  activity  of  the  virus  itself.  After  one  to  two  months' 
storage,  which  is  generally  practiced  to  reduce  the  number  of  bac- 
teria, the  vaccine  is  relatively  free  from  microorganisms,  although 
it  is  practically  never  sterile. 

The  ripened  vaccine  is  subjected  to  a  bacteriological  examination 
to  determine  the  number  of  bacteria  per  cubic  centimeter,  the  absence 
of  tetanus  bacilli  and  streptococci,  and  a  guinea-pig  inoculation  is 
is  made  with  about  a  cubic  centimeter  of  it  to  guard  against  an  acci- 
dental excess  of  carbolic  acid,  before  it  is  tested  clinically  for  its 
potency.  The  potency  test  is  made  upon  several  children  (previously 
unvaccinated)  in  the  usual  manner.  Generally  at  least  a  dozen  cases 
are  vaccinated  and  a  high  percentage  of  "takes"  must  be  obtained 
before  the  product  is  finally  marketed. 

Recently  Noguchi1  has  cultivated  an  absolutely  sterile  vaccine  virus 
of  high  potency  in  the  testes  of  rabbits  and  bulls.  The  entire  freedom 
of  the  preparation  from  alien  microorganisms  not  only  eliminates  the 
necessity  of  a  ripening  process  to  reduce  bacterial  contamination; 
it  also  makes  it  possible  to  reduce  the  cost  of  production  materially. 
The  vaccinal  eruption  induced  in  the  cornea,  skin  and  testes  of  rabbits 
and  the  skin  eruptions  in  calves  were  identical  with  those  induced 
by  the  virus  perpetuated  in  the  ordinary  manner.  The  eruptions 
induced  in  man  also  were  perfectly  typical.  Finally,  the  sterile  tes- 
ticular  vaccine  induced  immunity  reactions  in  experimental  animals 
identical  with  those  obtained  with  the  ordinary  ".skin"  vaccine. 

Phenomena  of  Vaccination. — 1.  Technic. — The  site  of  vaccination, 
preferably  the  outer  aspect  of  the  arm  about  the  deltoid  muscle,  is 
cleansed  thoroughly  with  soap  and  water,  and  finally  with  alcohol 
if  possible.  When  the  surface  is  dry  a  light  scratch  about  an  inch 
long  is  made  with  a  sterile  needle,2  deep  enough  so  that  the  bottom 
of  the  incision  is  slightly  reddened,  but  not  deep  enough  to  draw  blood. 
The  virus  is  then  spread  over  the  area  and  brought  into  intimate 
contact  with  the  epidermal  layer  by  gentle  rubbing  with  the  side  of 
the  needle.  The  safest  method  of  vaccination  is  by  puncture  either 
with  a  charged  needle,  or  through  a  shallow  abrasion  made  with  a 
von  Pirquet  tuberculin  chisel.  The  chances  of  successful  vaccination 
by  the  puncture  method  are  much  less  than  by  the  linear  incision, 
however.  The  older  method  of  vaccination  was  through  a  scarified 

1  Jour.  Exp.  Med.,  1915,  xxi,  539. 

2  An  ordinary  sewing  needle  is  excellent  for  the  purpose. 


SMALLPOX  AND   VACCINIA  575 

area,  varying  from  a  square  centimeter  to  nearly  twice  that  size.  The 
crust  that  forms  over  such  a  wound  furnishes  excellent  anaerobic 
conditions  for  the  growth  of  bacteria,  and  the  thickness  of  the  crust 
offers  mechanical  opposition  to  the  formation  of  the  vesicles,  which 
are  prone  to  appear  around  the  area  in  consequence.  Vaccination  by 
scarification  is  forbidden  by  law  in  Germany. 

2.  The  Course  of  the  Disease,  Vaccinia. — The  initial  reddened  site 
of  inoculation  soon  disappears,  leaving  only  a  small  scratch  or  punc- 
ture; about  the  third  or  fourth  day,  however,  one  or  several  small 
bright  red  papules  appear,  which  become  vesicular  by  the  end  of 
seven  days  and  surrounded  with  a  bright  red  areola.     The  contents 
of  the  vesicle  become  yellowish,  usually  from  the  eighth  to  the  tenth 
day,  and  discharge  a  yellowish  fluid  if  they  are  opened.    The  contents 
then  become  dessicated,  and  a  crust  forms  which  drops  off  in  about 
two  weeks. 

From  the  third  to  the  fifth  day  after  the  vaccination  a  febrile 
reaction  of  one  or  two  degrees  is  usually  experienced,  and  the  site  of 
the  vaccination  itches  intensely  and  is  painful.  There  is  frequently 
loss  of  appetite  and  general  symptoms  of  malaise  quite  out  of  propor- 
tion to  the  amount  of  local  reaction.  By  the  end  of  the  second  week  the 
symptoms  have  disappeared  and  the  sunken  multilocular  scar  is  the 
principal  residual  evidence  of  a  successful  vaccination.  It  is  generally 
believed  that  already  by  the  ninth  to  the  eleventh  day  after  inoculation 
the  patient  is  relatively  refractory  to  infection  with  smallpox  virus. 

3.  Immunity. — The  duration  of  immunity  is  not  definitely  known, 
but  it  is  stated  to  be  from  seven  to  ten  years  on  the  average.    In  Ger- 
many, where  vaccination  has  been  enforced  by  law  for  five  decades, 
a  child  is  required  to  be  vaccinated  by  the  end  of  the  first  year,  again 
about  the  time  it  enters  school,  and  a  third  time  at  the  age  of  sixteen 
or  thereabouts. 

Occasionally  a  first  vaccination  is  unsuccessful.  Frequently  old  or 
inactive  vaccine,  poor  technic,  or  a  deliberate  sterilization  of  the 
vaccined  area  with  disinfectants  are  responsible,  because  man  does 
not,  as  a  rule,  exhibit  immunity  to  natural  vaccinia.  Several  suc- 
cessive negative  results  should  be  obtained  before  the  individual  is 
pronounced  refractory. 

4.  Revaccination. — Revaccination    frequently   does   not   lead   to   a 
"take,"  but  in  a  fair  proportion  of  individuals  a  typical  reaction  may 
take  place;  this  may  be  an  accelerated  reaction.     The  accelerated 
reaction  runs  a  more  rapid  course  than  the  ordinary  reaction  and 


576  DISEASES  OF   UNKNOWN  ETIOLOGY 

reaches  maturity  usually  within  four  to  six  days  in  place  of  seven  to 
ten  days.  Less  commonly  an  "immediate"  reaction  is  met  with; 
the  site  of  inoculation  becomes  reddened  and  the  lesion  is  greatest 
within  twenty-four  hours  after  the  inoculation.  The  reddened  area 
fades  rapidly  and  the  entire  process  heals  almost  as  quickly  as  the 
simple  reaction  of  trauma  excited  by  the  scratch  in  the  epidermis. 
The  accelerated  and  immediate  reactions  are  usually  regarded  as 
potentially  equivalent  to  a  typical  reaction,  provided  they'  are  induced 
by  re  vaccination. 

Dengue.-  r  The  etiology  of  dengue  has  not  been  definitely  estab- 
lished, but  it  appears  to  belong  to  the  group  of  filterable  viruses  and 
to  be  transmitted  by  Culex  fatigans,  a  mosquito  very  common  in  the 
tropics.  Graham1  claimed  to  have  transmitted  the  disease  to  non- 
immune  individuals  not  only  through  the  bite  of  infected  female 
Culices,  but  also  by  injecting  the  ground  up  salivary  glands  of  a  mos- 
quito that  had  previously  bitten  a  patient.  Ashburn  and  Craig2 
state  that  the  virus  will  pass  a  Berkefeld  filter  and  that  both  whole 
blood  and  serum  filtered  through  Berkefeld  filters  will  reproduce  the 
disease  in  non-immune  individuals.  The  incubation  period  in  these 
cases  was  about  four  days.  Ashburn  and  Craig  believe  with  Graham 
that  the  virus  of  dengue  is  ordinarily  transmitted  by  Culex  fatigans. 

Rocky  Mountain  Spotted  Fever. — Rocky  Mountain  Spotted  Fever 
is  an  acute  fever  characterized  by  a  purpuric  eruption  of  the  skin. 
The  disease  is  rather  strictly  limited  to  the  Northern  Rocky  Mountain 
States,  Montana,  Wyoming  and  Idaho. 

The  etiological  agent  is  not  definitely  known.  Wilson  and  Chown- 
ing3  believed  the  causative  agent  to  be  a  Babesia  transmissible  by  a 
tick,  Dermacentor  reticularis  (now  known  as  Dermacentor  occiden- 
talis).  This  view  was  not  supported  by  later  observers.  Ricketts4 
in  numerous  investigations  has  shown  that  the  virus  circulates  in  the 
blood  stream,  and  that  infected  ticks  may  transmit  the  disease.  He 
was  also  successful  in  infecting  monkeys  (Macacus  rhesus)  and  guinea- 
pigs  with  the  virus.  One  attack  conferred  immunity  to  subsequent 
infection  in  experimental  animals,  and  the  serum  of  an  immune 
animal  protected  a  susceptible  animal  from  infection.  As  a  curative 
agent  the  serum  was  of  little  value.  A  minute  diplococcoid  or  bipolar 

1  Jour.  Trop.  Med.,  1903,  vi,  209. 

2  Philippine  Jour.  Sci.,  1907,  ii,  93. 

3  Jour.  Inf.  Dis.,  1904,  i,  31. 

4  Jour.  Am.  Med.  Assn.,  1906,  xlvii,  33,  358;    1907,  xlix,  24,  1278;    Trans.  Chicago 
Path,  Soc.,  1907;    Jour.  Inf.  Dis.,  1908,  v,  221;    Jour.  Am.  Med.  Assn.,  1909,  lii,  379. 


MUMPS  577 

staining  structure  was  observed  in  great  numbers  in  the  blood  of 
infected  men  and  in  the  eggs  of  infected  ticks.  These  were  not  suc- 
cessfully cultivated,  but  agglutinated  with  the  serum  of  an  immune 
animal.  Their  relation  to  the  disease  has  not  been  established. 

Mumps. — Mumps  or  epidemic  parotitis  is  a  specific  infectious 
disease  which  is  more  commonly  observed  among  children  from  four 
to  fifteen  years  of  age;  although  younger  children  and  adults  are  by 
no  means  immune.  The  incubation  period  averages  from  seventeen 
to  twenty-eight  days.  It  is  probable  that  the  infectious  period  begins 
a  few  days — about  four — before  the  characteristic  syndrome  appears, 
and  the  disease  is  probably  transmitted  directly  from  person  to  per- 
son through  infected  material  from  the  nasopharynx.  The  mortality 
is  very  low  and  cases  that  terminate  fatally  are  generally  very  young 
children  and  infants. 

The  causative  agent  is  not  definitely  known :  a  diplococcus  has  been 
isolated  from  inflamed  parotid  glands  by  Laveran  and  Catrin1-  in 
sixty-seven  out  of  a  total  of  ninety-two  cases.  Mecray  and  Walsh,2 
Michaelis  and  Bienn,3  Busquet  and  Feri4  have  made  similar  isolations. 
Teissier  and  Esmein5  report  the  successful  culture  of  a  similar  organism 
from  a  case  of  suppurative  parotitis.  Herb6  has  also  isolated  a  diplo- 
coccus from  a  case  of  suppurative  parotitis  which  ended  fatally.  Ani- 
mal experiments  with  these  cultures  have  not  been  convincingly  posi- 
tive. 

Nicolle  and  Conseille7  and  Gordon,8  working  independently,  state 
that  fluid  separated  from  the  parotid  glands  of  patients  having  mumps, 
injected  into  the  parotid  glands  of  monkeys,  reproduced  a  syndrome 
strikingly  like  that  of  mumps  in  these  animals.  Gordon  also  found 
that  the  virus  retained  its  virulence  after  passage  through  a  Berkefeld 
filter.  It  is  destroyed  by  a  brief  exposure  to  55°  C.  It  would  appear 
from  his  observations  that  the  virus  of  mumps  belongs  to  the  group 
of  filterable  viruses. 

1  Compt.  rend.,  Soc.  biol.,  1893,  9  ser.,  v,  528. 

2  Medical  Record,  1896,  i,  440. 

a  Verhandl.  XV  Kongress  f.  inn.  Med.,  1897,  xv,  441. 

4  Rev.  d.  Med.,  1896,  xvi,  744. 

5  Compt.  rend.  Soc.  biol.,  1906,  Ix,  803,  853. 
e  Arch.  Int.  Med.,  1909,  iv,  201. 

7  Compt.  rend.  Acad.  sc.,  1913,  clvii,  340. 

8  Lancet,  1913,  ii,  275. 


37 


SECTION  IV. 

GASTRO-INTESTINAL  BACTERIOLOGY. 


CHAPTER  XXX. 

GASTRO-INTESTINAL  BACTERIOLOGY. 

General  Considerations. — An  examination  of  the  feces1  of  a  healthy 
adult  with  the  higher  objectives  of  the  microscope  will  show  that  a 
large  portion  of  the  fecal  mass  is  made  up  of  bacterial  cells.  An 
average-sized  bacterial  cell  is  very  small  indeed,  measuring  about 
1  micron  in  diameter  and  2  microns  in  length,  hence  it  is  not  surprising 
that  various  investigators  have  estimated  the  daily  excretion  of  bac- 
teria by  a  healthy  adult  on  a  mixed  diet  at  one  hundred  to  thirty-three 
hundred  billions.  The  bacteria  when  dried  would  weigh  more  than 
5  grams  and  would  contain  about  0.6  grams  of  nitrogen.  A  very 
considerable  proportion  of  the  total  nitrogen  of  the  feces  is  contained 
in  these  bacteria. 

It  is  apparent  that  the  ingested  food  does  not  contain  this  prodigious 
number  of  bacteria,  consequently  it  must  be  assumed  that  there  is 
a  rapid  development  of  the  organisms  in  the  intestinal  tract.  The 
theoretical  progeny  of  a  single  bacterial  cell  of  the  more  rapidly 
developing  types  may  number  millions  in  twenty-four  hours,  so  that 
the  mechanical  possibility  of  a  very  great  daily  proliferation  of  bacteria 
is  well  established.  It  is  obvious,  therefore,  that  the  alimentary  canal, 
from  the  viewpoint  of  bacteriology,  is  a  most  efficient  incubator  and 
cultural  medium  combined,  in  which  bacterial  growth  exceeds  both  in 
intensity  and  complexity,  that  of  any  known  medium.  The  range  of 
reaction  and  composition  of  nutritive  substances  at  different  levels 
of  the  intestinal  tract  are  such  that  theoretically  a  great  variety  of 
bacteria  capable  of  developing  at  body  temperature  may  find  condi- 
tions favorable  for  their  growth  there.2  The  prominent  types  of 

1  Average  weight  100  to  200  grams  per  diem. 

2  Kendall,  Jour.  Biol.  Chem.,  1909,  vi,  499;  Wisconsin  Med.  Jour.,  1913,  xii,  No.  1. 


580  GASTRO-INTESTINAL  BACTERIOLOGY 

bacteria  that  appear  in  the  intestinal  flora  of  a  normal  person  are 
fairly  constant  in  their  occurrence,  but  there  may  be  well-marked 
seasonal  and  even  annual  variations  in  the  relative  proportions  of  the 
individual  groups  of  organisms  which  comprise  this  flora.  This  sug- 
gests that  the  normal  bacterial  flora  is  acclimatized  to  the  intestinal 
environmental  conditions  of  temperature,  reaction  and  composition 
of  food,  and  of  intestinal  secretions  at  different  levels.  It  also  indi- 
cates that  the  activities  of  the  organisms  which  comprise  the  normal 
intestinal  flora  are  not  in  active  opposition  to  those  of  the  host.1 

Adventitious  bacteria,  frequently  in  considerable  numbers, 
undoubtedly  reach  the  intestinal  tract  from  time  to  time.  The  fate 
of  these  organisms  depends  upon  a  number  of  factors,  some  of  which 
are  little  understood.  If  their  activities  are  greatly  at  variance  with 
those  of  the  normal  types  they  usually  fail  to  gain  a  foothold;  either 
they  are  unable  to  develop  in  competition  with  the  well-acclimatized 
normal  flora,  or  they  cannot  accommodate  themselves  to  the  physio- 
logical and  chemical  conditions  which  prevail  there.  If,  on  the  con- 
trary, these  organisms  can  adapt  themselves  readily  to  the  prevailing 
conditions  at  some  level  of  the  alimentary  canal  they  may  continue 
to  develop  either  in  association  with  preexisting  types,  or  gradually 
replace  the  latter.2  It  is  doubtless  through  this  process  that  the  sea- 
sonal prevalence  of  some  types  of  intestinal  bacteria  has  its  origin. 
It  is  not  unlikely,  furthermore,  that  the  occasional  unusual  type  of 
organism  characteristic  for  an  individual  or  a  group  of  individuals 
gains  entrance  to  and  develops  in  the  intestinal  tract  in  this  manner. 

The  nature  of  the  process  whereby  progressively  pathogenic  bacteria 
(usually  of  exogenous  origin)  replace  or  modify  the  normal  intestinal 
flora  is  as  yet  little  understood.  There  is  evidence  in  favor  of  the  view 
that  exogenous  bacteria  which  invade  the  body  through  the  intestinal 

1  The  general  phenomena  governing  the  parasitism  of  bacteria  in  the  alimentary  canal 
are  not  unlike  those  leading  to  bacterial  parasitism  upon  the  skin,  the  conjunctiva, 
or  other  surfaces  of  the  body  which  are  in  communication  with  the  exterior.     One 
important  phase  of  intestinal  parasitism  is  not  manifested  in  other  parts  of  the  body, 
however.     The  bacteria  of  the  intestinal  flora  change  along  rather  definite  lines  from 
infancy  to  adult  life,  as  the  diet  of  the  host  changes  from  the  monotonous  pabulum  of 
infancy  to  the  varied  regimen  of  the  adult.     The  organisms  parasitic  upon  the  skin  and 
other  surfaces  of  the  body  do  not  exhibit  this  change  in  type,  and  it  is  reasonable  to 
attribute  the  relative  stability  of  the  skin  flora  to  the  relative  constancy  of  environ- 
mental conditions  there,  while  the  succession  of  types  of  intestinal  bacteria  from  infancy 
to  adult  life  is  rather  definitely  associated  with  corresponding  changes  in  the  diet  of  the 
host. 

2  Undoubtedly  repeated  inoculation  of  the  alimentary  canal  with  adventitious  strains 
of  bacteria  plays  an  important  part  in  determining  their  acclimatization  in  the  intestines; 
possibly  a  simultaneous  absence  of  the  preexisting  intestinal  types  in  the  environment, 
leading  to  a  reduction  or  even  absence  of  these  normal  inhabitants  in  the  food  of  the 
host  may  materially  affect  the  outcome  of  the  "replacement"  process. 


THE  GASTRO-INTESTINAL  FLORA  OF  NORMAL  INFANTS     581 

tract  may  become  somewhat  widely  disseminated  in  restricted  areas 
and  appear  in  the  intestinal  contents  of  many  individuals  without 
inciting  noteworthy  symptoms,  prior  to  the  appearance  of  disease  in 
epidemic  proportions1  and  with  characteristic  symptoms. 

THE    GASTRO-INTESTINAL   FLORA    OF   NORMAL   INFANTS, 
ADOLESCENTS   AND   ADULTS. 

The  fetal  intestinal  contents,  the  meconium,  are  sterile  at  birth; 
the  first  bacteria  appear  in  the  meconium  from  eighteen  to  twenty- 
four  hours  postpartum.  This  is  a  period  of  adventitious  infection 
during  which  a  variety  of  bacterial  types,  largely  determined  by  the 
environment  of  the  infant,  gain  entrance  to  the  alimentary  canal  by 
way  of  the  mouth  or  anus  and  are  excreted  in  the  residual  embryonic 
feces.  This  initial  non-characteristic  intestinal  flora  is  usually  more 
varied  in  summer  than  in  winter  and  more  luxuriant  when  the  infant 
is  exposed  to  relatively  uncleanly  surroundings  than  when  the  reverse 
is  the  case.  Escherich2  and  others  have  called  attention  to  the  occur- 
rence of  a  rather  large  bacillus  in  the  meconium,  possessing  a  terminal 
spore  closely  resembling  Bacillus  tetani.  This  organism,  known  as  the 
Kopfchen  bacillus,  has  been  identified  by  some  observers  as  Bacillus 
putrificus  of  Bienstock;3  it  has  not  been  studied  culturally,  however, 
and  this  identification  cannot  be  regarded  as  final.  Other  spore- 
forming  bacteria,  both  aerobic  and  anaerobic,  are  also  usually  present 
in  the  meconium  at  this  period.  Of  these  Bacillus  aerogenes  capsulatus 
and  members  of  the  Bacillus  Mesentericus  Group  are  the  best  known. 
Bacillus  coli,  Bacillus  proteus,  Bacillus  lactis  aerogenes  and  Micro- 
coccus  ovalis4  also  occur  commonly. 

The  initial  period  of  adventitious  bacterial  infection  of  the  intestinal 
contents  merges  more  or  less  imperceptibly  through  a  transitional 
stage  to  the  period  of  dominance  of  the  characteristic  infantile  intes- 
tinal flora,  which  becomes  settled  usually  about  the  third  day  post- 
partum. At  this  time  the  breast  milk  diet  of  the  nursling  is  well 
established  and  the  intestinal  tract  is  permeated  with  it.  The  bacteria 
throughout  the  alimentary  canal  become  more  numerous,  the  spore- 


1  Kendall,  Boston  Med.  and  Surg.  Jour.,  1915,  clxxii,  851. 

2  Escherich,  Darmbakterien  des  Saiiglings,  Stuttgart,  1886,  p.  9. 

3  Arch.  f.  Hyg.,  1899,  xxxvi,  335;   ibid.,  1900,  xxix,  390. 

4  Micrococcus  ovalis  (Escherich,  loc.   cit.,  p.  89)   appears  to  be  identical  with  the 
enterocoque  of  the  French  writers,  with  Streptococcus  lacticus  of  Kruse  (Centralbl.  f. 
Bakt.,  Orig.,  1903,  xxxiv,  737)  and  Streptococcus  enteriditis  of  Hirsch  (ibid.,  1897,  xxii, 
369),  and  Libman  (ibid.,  1897,  xxii,  376). 


582  GASTRO-INTESTINAL  BACTERIOLOGY 

forming  types  disappear  for  the  most  part  and  rather  abruptly,  and 
the  coccal  forms  and  Gram-negative  bacilli  of  the  colon  aerogenes  type 
diminish  relatively,  but  never  quite  disappear.  Simultaneously  rather 
long,  thin  bacilli,  occurring  singly,  in  pairs,  or  in  groups  with  their 
axes  parallel,  become  strikingly  prominent.  These  bacilli  are  fre- 
quently slightly  curved  and  occasionally  their  ends  are  somewhat 
attenuated.  Typically  they  are  Gram-positive  and  stain  uniformly, 
but  in  many  instances  they  exhibit  a  central  Gram-positive  granule 
in  an  otherwise  Gram-negative  rod,  presenting  the  "punctate"  appear- 
ance described  by  Escherich.1  Occasionally  the  cytoplasm  of  these 
organisms  is  collected  into  small,  round  or  oval  granules  which  stain 
intensely;  the  remainder  of  the  rod  stains  faintly  or  not  at  all.  At 


FIG.  96. — Bacillus  bifidus.     Sediment  from  lactose  fermentation  tube.    X  1000. 

first  sight  these  granules  resemble  chains  of  cocci.  This  somewhat 
pleiomorphic  organism  is  Bacillus  bifidus,  first  observed  by  Escherich, 
but  isolated  in  pure  culture  and  studied  in  detail  by  Tissier.2  It  is  an 
obligate  anaerobe,3  fermentative  in  character,  which  typically  forms 
considerable  amounts  of  acid  from  lactose  and  other  sugars,  but  no 
gas.  The  organism  received  the  name  "bifidus"  from  its  remarkable 
property  of  developing  well-defined  bifid  ends  when  it  is  grown  in 
artificial  media;  it  does  not  ordinarily  exhibit  bifid  ends  in  the  intes- 
tinal tract.  Moro4  and,  independently,  Finkelstein5  have  isolated  and 
described  an  organism  very  similar  in  morphology  to  Bacillus  bifidus 

1  Loc.  cit. 

2  Recherches  sur  la  Flore  Intestinale  des  Nourrissons,  etc.,  These  de  Paris,  1900,  p.  85. 

3  Noguchi  (Jour.  Exp.  Med.,  1910,  xii,  182)  appears  to  have  shown  that  Bacillus  bifidus, 
under  laboratory  conditions,  may  become  aerobic  and  form  spores. 

4  Wien.  klin.  Wchnschr.,  1900,  xiii,  114. 

6  Deutsch.  med.  Wchnschr.,  1900,  xxii,  263. 


THE  GASTRO-INTESTINAL  FLORA  Of  NORMAL  INFANTS     583 

as  it  occurs  in  the  intestinal  contents,  but  which  differs  materially 
from  the  latter  both  in  its  aerobiosis  and  in  its  inability  to  develop 
bifid  ends  in  artificial  media.  This  organism,  Bacillus  acidophilus, 
is  more  commonly  found  in  the  intestinal  contents  of  artificially  fed 
babies  than  in  nurslings,  and  it  is  more  tolerant  of  organic  acids  than 
Bacillus  bifidus.  It  belongs  to  the  group  of  Aciduric  Bacteria.1 

In  addition  to  Bacillus  bifidus2  and  Bacillus  acidophilus,  which 
typically  comprise  a  majority  of  the  characteristic  intestinal  bacteria, 
smaller  numbers  of  Micrococcus  ovalis,  Bacillus  coli,  Bacillus  lactis 
aerogenes  and  other  bacteria  are  found  in  the  feces  of  nurslings. 

Escherich3  has  emphasized  the  very  significant  fact  that  putrefactive 
(proteolytic)  bacteria  are  uncommon  in  the  dejecta  of  normal  nurs- 
lings; there  is  little  or  no  evidence  of  the  development  of  these 
organisms  in  the  intestinal  tract  during  this  stage.  The  putrefactive 
bacteria,  as  a  rule,  do  not  develop  in  an  acid  medium  in  competition 
with  organisms  like  Bacillus  bifidus  and  other  acidogenic  types  which 
dominate  the  alimentary  canal  of  the  normal  nursling. 

Distribution  of  the  Intestinal  Flora  of  the  Normal  Nursling. — The 
principal  portal  of  entry  of  the  intestinal  bacteria  is  the  mouth.  There 
is'  no  doubt  that  a  great  variety  of  organisms  may  from  time  to  time 
enter  this  atrium,  including  not  only  the  ordinary  organisms  of  the 
nurslings'  environment,  but  pathogenic  bacteria  as  well.  A  majority 
of  these  pass  to  the  stomach,  and  they  may  pass  to  the  intestinal 
tract.  The  flora  of  the  mouth  and  stomach  are  not^well  known,  but 
they  appear  to  be  of  relatively  slight  importance  as  a  rule.  Those 
adventitious  organisms  which  pass  from  the  stomach  to  the  duodenum 
rarely  appear  to  gain  a  foothold  there,  or  at  lower  levels  of  the 
intestines. 

The  duodenal  flora,  which  in  health  is  composed  chiefly  of  coccal 
forms  of  the  Micrococcus  ovalis  type,  is  most  numerous  during  those 
periods  when  the  food  is  passing  through;  during  interdigestive  periods 
there  appear  to  be  relatively  few  bacteria  at  this  level.  From  the 
jejunum  to  the  ileocecal  valve,  members  of  the  Bacillus  lactis  aerogenes 
group  occur  more  commonly.  Bacillus  coli  and  other  members  of  the 
colon  group  are  most  numerous  at  the  ileocecal  valve  and  the  cecum, 

1  Kendall,  Jour.  Med.  Research,  1910,  xxii,  153;  Rahe,  Jour.  Inf.  Dis.,  1914,  xv,  141. 

2  Madame  Tsiklinsky  (Ann.  Inst.  Past.,  1903,  xvii,  317)  has  been  unable  to  demon- 
strate B.  bifidus  in  normal  nurslings'  feces  as  frequently  as  has  been  reported  elsewhere; 
the  consensus  of  opinion  appears  to  be,  however,  that  bifidi  are  the  most  characteristic 
bacilli  of  the  normal  nursling  intestinal  flora. 

3  Loc.  cit. 


584  G ASTRO-INTESTINAL  BACTERIOLOGY 

and  Bacillus  bifidus  or  similar  organisms  dominate  the  large  intestines 
from  this  level  to  the  sigmoid  flexure.  The  remainder  of  the  large 
intestine  to  the  rectum  is  somewhat  sparsely  populated  with  living 
bacteria,  partly  because  the  fecal  mass  is  relatively  desiccated  by  the 
absorption  of  water,  partly  because  of  the  accumulation  of  waste 
products  of  bacterial  activity — principally  acids  resulting  from 
fermentation  of  lactose,  formed  higher  up  in  the  tract — which  inhibit 
the  development  of  bacteria  in  the  lower  levels.1 

It  must  be  remembered  that  while  the  greatest  number  of  impor- 
tant bacteria  mentioned  above  occur  at  the  levels  indicated,  there  is 
a  mechanical  transportation  of  all  intestinal  bacteria  from  the  higher 
to  the  lower  levels,  so  that  some  organisms  of  all  types  are  found  in 
the  dejecta.  It  is  particularly  important  to  realize  that  the  types 
of  bacteria  outlined  are  those  which  can  be  identified  by  staining 
methods  as  numerically  prominent  at  the  various  intestinal  levels; 
these  observations  can  be  corroborated  by  appropriate  cultural 
methods.  Nevertheless,  there  is  a  wide  disproportion  between  the 
numbers  of  each  of  the  respective  bacteria  seen  in  stained  preparations 
and  the  numbers  of  each  type  which  develop  in  artificial  media.  Thus, 
Escherich2  observed  that  a  preponderance  of  bacteria  of  normal  nurs- 
lings' feces  were  Gram-positive  bacilli,  yet  he  never  succeeded  in  grow- 
ing these  bacilli  in  artificial  media;  the  principal  types  which  developed 
in  his  cultures  were  Bacillus  coli  and  Bacillus  lactis  aerogenes,  organ- 
isms which  are  numerically  in  the  minority  in  the  intestines,  but  which 
grow  luxuriantly  outside  the  body.  It  is  now  realized  that  he  did 
not  employ  suitable  conditions  of  culture  to  isolate  the  most  prominent 
types  of  organisms.  Undoubtedly  much  of  the  confusion  which  has 
attended  the  study  of  intestinal  bacteriology  in  the  past  is  attributable 
to  the  lack  of  appreciation  of  the  cultural  peculiarities  of -the  intestinal 
organisms. 

Distribution  of  the  Intestinal  Flora  of  Artificially  Fed  Infants.— 
Escherich3  directed  attention  to  the  striking  dissimilarity  between  the 
intestinal  flora  of  the  breast-fed  and  the  artificially  fed  infant;  cul- 
turally, morphologically  and  chemically  the  former  is  more  homogen- 
eous than  the  latter.  The  most  distinctive  features  of  the  dejecta 
of  artificially  fed  infants  are:  the  relative  increase  of  Gram-negative 
bacteria  of  the  coli-aerogenes  type,  and  of  coccal  forms  of  the  Micro- 
coccus  ovalis  type,  together  with  a  diminution  of  Bacillus  bifidus. 

1  Kendall,  Jour.  Med.  Research,  1911,  xxv,  117,  et  seq. 

2  Loc.  cit.  3  Loc.  cit. 


THE  G ASTRO-INTESTINAL  FLORA   OF  NORMAL  INFANTS     585 

Bacillus  acidophilus  is  relatively  more  numerous,  as  a  rule,  in  the 
artificially  fed  infant  than  in  the  nursling.  Proteolytic  bacteria  of 
several  types  are  also  of  frequent  occurrence,1  but  they  are  not  com- 
monly found  in  the  dejecta  of  normal  nurslings.  These  organisms  are 
frequently  spore-forming  bacilli,  of  which  two  principal  groups  are 
recognized — members  of  the  aerobic  group,  of  which  Bacillus  mesen- 
tericus  is  a  prominent  type,  and  anaerobic  bacteria;  of  the  latter, 
Bacillus  aerbgenes  capsulatus  is  most  widely  known;  it  frequently 
occurs  in  small  numbers  in  the  feces  of  artificially  fed  infants.2  The 
reaction  of  normal  feces  of  artificially  fed  babies  is  usually  alkaline; 
culturally  and  chemically,  the  evidence  of  intestinal  proteolysis  of 
bacterial  causation  is  more  marked  in  these  infants  than  in  normal 
nurslings. 

The  general  distribution  of  types  of  bacteria  at  the  different  levels 
of  the  intestinal  tract  is  similar  to  that  observed  in  normal  nurslings; 
the  principal  differences  are  found  in  the  cecum  and  large  intestine, 
where  the  obligately  fermentative  bacteria  of  the  bifidus  type  are 
replaced  to  a  considerable  degree  by  an  extension  of  the  habitat  of 
the  colon  bacillus,  of  Bacillus  acidophilus,  and  the  appearance  of 
moderate  numbers  of  proteolytic  bacteria,  both  aerobic  and  anaerobic; 
many  of  the  latter  are  sporogenic. 

The  prevailing  bacteria  of  the  artificially  fed  infant  may  be  changed 
along  fairly  definite  lines  by  varying  the  proportion  of  protein  to 
carbohydrate  in  the  diet,  and  by  substituting  one  carbohydrate  for 
another.  Thus,  a  continued  preponderance  of  protein  leads  to  a  partial 
or  even  practically  complete  suppression  of  the  activity  of  the 
bifidus-acidophilus  group,  and  a  noteworthy  increase  in  the  activity 
of  proteolytic  organisms;3  of  the  latter,  aerogenic  bacteria  of  the 
colon-proteus  group  and  spore-forming  bacteria  of  the  mesentericus 
group  appear  to  be  the  more  prominent.  A  relative  increase  in  carbo- 
hydrate leads  to  a  diminution  or  suppression  of  proteolytic  activity 
in  the  intestinal  tract,  and  an  increase  in  the  fermentative  activities 
of  the  intestinal  organisms.4  Those  bacteria — as  Bacillus  coli — which 

1  Escherich,  loc.  cit. 

2  See  Hibler  (Untersuchungen  liber  die  pathogenen  Anaeroben,  Jena,  1908),  Jungano 
and  Distaso  (Les  Anaerobies,  Paris,  1910)  for  description  of  various  intestinal  anaerobes. 
Unfortunately,  so  little  is  definitely  known  about  a  majority  of  these  organisms,  cultur- 
ally, chemically  and  numerically,  that  almost  nothing  can  be  said  of  their  importance. 

3  Kendall,  Jour.  Biol.  Chem.,  1909,  vi,  268;  Herter  and  Kendall,  ibid.,  1910,  vii,  203. 

4  Provided,  of  course,  the  digestion  of  the  infant  remains  normal.     It  is  obvious  that 
a  disturbance  of  the  digestive  function  of  the  alimentary  canal  may  lead  to  new  factors 
which  may  play  an  important  part  in  determining  the  prevalence  of  one  or  several  types 
of  intestinal  bacteria. 


586  GASTRO-INTESTINAL  BACTERIOLOGY 

can  accommodate  their  metabolism  to  either  a  protein  or  carbo- 
hydrate regimen  become  fermentative  and  produce  lactic  acid  and 
other  products  of  the  fermentation  of  carbohydrate  in  place  of  H2S  and 
NH3,  indol,  and  other  putrefactive  products  which  characterize  their 
development  in  protein  media1  under  these  conditions.  The  obligately 
proteolytic  organisms  tend  to  decrease  in  number  because  they  are 
unable  to  thrive  in  the  presence  of  active  fermentation,  and  the 
carbohydrophilic  bacteria  increase  both  in  numbers  and  in  activity; 
the  type  of  carbohydrophilic  organisms  which  develops  depends  upon 
the  carbohydrate  fed  and  upon  the  length  of  time  the  diet  is  con- 
tinued; Bacillus  bifidus  tends  to  increase  in  numbers2  when-lactose  is 
the  sugar,  Bacillus  acidophilus  if  maltose  is  substituted  for  lactose, 
provided  the  regimen  is  maintained  for  several  days.3 

The  changes  in  the  intestinal  flora  from  the  bottle-fed  infant  to 
adolescence  and  adult  life  depend  somewhat  upon  the  diet  of  the 
individual.  The  general  tendency  in  individuals  on  an  average  mixed 
diet  is  for  Bacillus  coli  to  become  the  dominating  organism;  usually 
about  75  per  cent,  of  the  viable  bacteria  of  the  feces  are  colon  bacilli. 
Of  the  remaining  organisms,  spore-forming  organisms  of  the  mesen- 
tericus  group  are  usually  numerous,  and  gas  bacilli  may  be  found 
relatively  frequently,  but  in  small  numbers.  Bacillus  coli  and  Bacillus 
mesentericus  are  among  the  most  persistent  of  the  intestinal  bacteria 
of  adults.  Those  two  organisms  and  no  others  were  found  in  the 
lower  part  of  the  large  intestine  of  a  man  who  abstained  from  all  food 
for  thirty-one  days.4  The  characteristic  feature  of  the  normal  adult 
fecal  flora  as  compared  with  the  infantile  nursling  flora  is  the  very 
heterogeneous  variety  of  types  of  bacteria  in  the  former,  in  sharp 
contrast  to  the  homogeneity  of  types  of  bacteria  in  the  latter. 

Distribution  of  the  Intestinal  Flora  in  the  Adolescent  and  Adult. 
The  stomach  in  health  is  quite  free  from  bacteria  as  a  rule.  It  has 
been  assumed  in  the  past  that  the  hydrochloric  acidity  may  be  a 
factor  in  the  destruction  of  organisms,  but  it  should  be  remembered 
that  protein  undergoing  gastric  digestion  binds  hydrochloric  acid. 
Nevertheless,  bacterial  activity  is  very  limited  in  the  stomach  under 
normal  conditions. 

1  Kendall,  Boston  Med.  and  Surg.  Jour.,  1910,  clxiii,  322;  Pediatrics,  1910,  xxii,  No.  9. 

2  It  is  apparent  that  this  change  cannot  take  place  unless  there  is  a  residuum  of  bifidi 
in  the  intestinal  tract  to  develop  from.     The  same  is  true  for  Bacillus  acidophilus.     In 
the  absence  of  these  types  the  dominant  fermenting  organisms  will  vary  with  the  flora 
of  the  individual. 

3  Kendall,  Boston  Med.  and  Surg.  Jour.,  1910,  clxiii,  322. 

4  Kendall,  Publication  203  of  the  Carnegie  Institution  of  Washington.  1915,  p.  232. 


THE  GASTRO-INTESTINAL  FLORA  OF  NORMAL  INFANTS     587 

The  duodenum  of  adults  is  relatively  poorly  populated  with  bac- 
teria in  interdigestive  periods,  and  Gushing  and  Livingston1  have 
called  attention  to  the  relative  innocuousness  of  gunshot  wounds  at 
this  level  as  contrasted  with  those  at  lower  levels,  where  peritonitis 
practically  invariably  follows  perforation  of  the  gut.  This  phenomenon 
is  not  wholly  attributable  to  the  comparative  paucity  of  bacteria  in 
the  duodenum  as  contrasted  to  lower  levels;  a  final  explanation  is 
lacking  at  the  present  time.  According  to  Gessner,2  staphylococci 
and  streptococci  are  numerous  in  the  duodenum,  and  Tavel  and  Lanz3 
have  made  similar  observations.  Recently  Hess,  using  a  duodenal 
catheter,4  has  studied  the  duodenal  flora  in  normal  individuals.  He 
finds  the  bacterial  content  very  low  in  interdigestive  periods;  staphy- 
lococci and  a  few  Gram-positive  and  Gram-negative  bacteria  were 
the  prevailing  types.  These  Gram-negative  bacteria  were  not  Bacillus 
coli.  Breast-fed  infants  showed  fewer  bacteria  in  the  duodenal  region 
than  did  bottle-fed  babies. 

The  lower  levels  of  the  small  intestines  become  progressively  richer 
in  bacteria.  The  relative  slowness  with  which  food  passes  through 
the  intestines  at  the  lower  levels  probably  is  a  potent  factor  in  creating 
conditions  favorable  for  continual  bacterial  growth.  As  a  rule  cocci 
still  predominate  in  the  lower  jejunum  and  upper  ileum,  but  Gram- 
negative  bacilli  of  the  colon  group  appear  in  moderate  numbers. 

The  cecum  and  ascending  colon  are  the  regions  of  most  intense  bac- 
terial proliferation  in  health,  but  the  number  of  living  bacteria  in  the 
intestinal  contents  diminishes  rather  abruptly  from  the  sigmoid  to  the 
rectum.  It  has  been  stated  that  at  least  90  per  cent,  of  the  bacteria 
of  the  feces  are  dead,  or  so  attenuated  in  vitality  that  they  are 
incapable  of  growing  in  artificial  media.  For  various  reasons  the 
accuracy  of  this  statement  may  be  questioned,  but  there  is  little  doubt 
that  the  numbers  of  viable  bacteria  in  the  relatively  desiccated  feces 
are  less  than  those  in  the  more  fluid  intestinal  contents  at  the  level 
of  the  cecum. 

The  bacteria  commonly  present  in  the  ileocecal  region  are  undoubt- 
edly of  many  and  varied  types,  but  in  general  aerogenic  bacilli  of  the 
colon  type5  (including  probably  members  of  the  proteus  group  as  well) 

1  Contributions  to  the  Science  of  Medicine  by  the  pupils  of  William  Welch,  1900,  543. 

2  Arch.  f.  Hyg.,  1889,  ix,  128.  3  Mitt.  a.  klin.  d.  Schweiz,  i. 

4  Ergebnisse  der  inn.  Med.  u.  Kinderheilk.,  1914,  xiii,  530. 

5  Ford,  Classification  and  Distribution  of  the  Intestinal  Bacteria  in  Man,  Studies  from 
the  Royal   Victoria  Hospital,  1903,  i,  No.  5;    MacConkey,  Jour.  Hyg.,  1905,  v,  333, 
have  described  the  common  types  of  aerobic  bacilli  in  the  intestinal  tract.     The  cultural 
characters   of   the  various   aerogenic  lactose-fermenting  organisms,   grouped   for  con- 
venience as  the  colon  group,  are  clearly  set  forth  in  these  monographs. 


588  GASTRO-INTESTINAL  BACTERIOLOGY 

and  aerobic  spore-forming  bacteria  of  the  mesentericus  group  are 
the  most  readily  recognized.  The  important  feature  of  the  intestinal 
flora  at  the  lower  levels  of  the  intestinal  tract  of  adolescents,  and 
more  especially  of  adults,  is  the  presence  of  facultative  fermentative 
bacteria  which  appear  to  thrive  equally  well  when  the  intestinal  con- 
tents at  this  level  contain  protein  and  carbohydrate  as  when  the 
carbohydrate  is  absent.  Members  of  the  colon-proteus  group,  par- 
ticularly the  former,  various  aerobic  liquefying  bacilli — both  spore- 
forming,  and  non-spore-forming — and,  to  a  limited  extent,  anaerobic 
bacteria  as  well  are  characteristic  of  the  bacterial  flora  of  the  large 
intestines  of  adults.  This  is  in  striking  contrast  to  the  distinctive 
monotonous  fermentative  flora  of  the  normal  nursling,  whose  diet 
contains  a  sufficient  amount  of  carbohydrate  (lactose)  to  bathe  the 
entire  alimentary  canal.  It  contrasts  also,  to  a  somewhat  lesser  degree, 
with  the  lower  intestinal  flora  of  young  children  on  a  cow's  milk  diet, 
where  the  proportion  of  carbohydrate  to  that  of  protein,  although 
decidedly  less  than  that  of  the  nursling,  is  usually  still  sufficient  to 
restrain  an  excessive  development  of  proteolytic  bacteria. 

It  will  be  seen  that  the  carbohydrate  of  the  infant  diet  is  lactose, 
which  is  utilizable  as  such  by  the  dominant  bacteria  of  the  infantile 
intestinal  and  fecal  flora.  A  not  inconsiderable  portion  of  the  carbo- 
hydrate of  the  adult,  on  the  contrary,  is  starch,  which  is  not  readily 
utilizable  as  such  by  a  great  majority  of  the  intestinal  or  fecal  bacteria; 
it  is  very  probable  that  a  very  considerable  proportion  of  the  assimil- 
able products  of  hydrolysis  of  the  starch  are  absorbed  rapidly  from  the 
intestinal  contents  and  therefore  there  is  normally  but  little  utilizable 
sugar  available  for  the  intestinal  flora  of  adults.  This  is  especially 
the  case  in  the  lower  levels  of  the  intestinal  tract,  where  the  stasis  of 
the  intestinal  contents  results  in  a  differential  accumulation  of  the 
more  slowly  hydrolyzed  and  absorbed  protein.  It  would  appear  from 
these  considerations  that  the  relative  absence  of  utilizable  carbo- 
hydrate in  the  large  intestine  of  adults  would  naturally  be  associated 
with  a  diminution  of  the  obligate  fermentative  or  carbohydrophilic 
organisms,  and  available  evidence  indicates  that  such  is  the  case. 

Significance  of  Intestinal  Bacteria. — The  striking  differences  in 
morphology,  chemistry  and  in  cultural  characters  between  the  intes- 
tinal floras  characteristic  respectively  of  nurslings,  artificially  fed 
infants  and  adults  suggest  at  once  that  nutritional  stimuli  may  be  an 
important  factor  in  determining  the  dominance  of  types  of  bacteria. 
An  intestinal 'flora  does  not  appear  to  be  essential  for  the  well-being 


THE   GASTRO-INTESTINAL  FLORA  OF  NORMAL  INFANTS     589 

of  mammals  in  the  Arctic  regions;  Levin1  has  found  that  the  feces 
of  polar  bears  are  practically  sterile.  It  must  be  remembered,  however, 
that  similar  animals  kept  in  captivity  in  more  temperate  climates 
exhibit  a  very  definite  intestinal  and  fecal  flora.  Attempts  to  rear 
chicks,2  turtles,3  tadpoles4  and  guinea-pigs5  in  a  sterile  environment 
have  not  added  materially  to  available  knowledge  of  the  physiological 
significance  of  the  intestinal  flora,  partly  because  the  rigorous  con- 
ditions under  which  such  observations  must  be  made  interfere  greatly 
with  the  normality  of  the  animals'  environment.  It  is  probable  that 
the  significance  of  the  intestinal  flora  lies  rather  in  its  potential  antag- 
onism to  alien  bacteria  which  certainly  gain  entrance  to  the  alimentary 
canal  from  time  to  time,  than  in  any  specific  participation  in  the 
normal  digestive  process  of  the  host.6 

The  normal  intestinal  flora  may  be  regarded  as  intestinal  parasites 
just  as  the  various  bacteria  which  occur  commonly  on  the  skin  are 
regarded  as  cutaneous  parasites.  It  is  important  to  realize  that  the 
normal  intestinal  organisms,  like  the  cutaneous  organisms,  are 
"opportunists,"  potentially  capable  of  becoming  invasive  whenever 
the  barriers  which  ordinarily  suffice  to  limit  their  development  to  the 
lumen  of  the  alimentary  canal  become  impaired,  giving  rise  to  endo- 
genous infections. 

Unlike  the  cutaneous  parasitic  flora  or  that  of  other  surfaces  of  the 
body  which  does  not  appear  to  vary  materially  from  infant  to  adult 
life,  the  intestinal  flora  changes  in  a  most  definite  and  striking  manner 
as  the  individual  develops  from  infancy  to  senescence.  This  change 
does  not  appear  to  depend  fundamentally  upon  bacteria  ingested 
with  the  food,  for  Escherich7  and  many  others  have  shown  that  steri- 
lization of  the  food  does  not  cause  a  noteworthy  reduction  in  the 
number  of  types  of  fecal  bacteria  in  young  children. 

The  most  important  normal  factor  in  determining  the  intestinal 

1  Ann.  Inst.  Past.,  1899,  xiii,  558;   Skandinavisches  Arch.  f.  Physiol.,  1904,  xvi,  249. 

2  Schottelius,  Arch.  f.  Hyg.,  1902,  xlii,  48. 

3  Moro,  Jahrb.  f.  Kinderheilk.,  1905,  xii,  467. 

4  Metchnikoff,  Ann.  Inst.  Past.,  1901,  xv,  361. 

5Nuttall   and   Thierfeldef,  Ztschr.  f.  physiol.  Chem.,  1895,  xxi,  109;  1896,  xxii,  62; 
1897,  xxiii,  231. 

6  Hilgermann  (Arch.  f.  Hyg.,  1905,  liv,  335)  and  others  have  produced  experimental 
evidence  in  favor  of  the  view  that  the  immature  intestinal  tract  of  the  young  infant  is 
more  permeable  to  bacteria  than  that  of  adolescents  and  adults.     It  may  be  inferred 
from  these  observations  that  the  normal  nursling  intestinal  flora  is  somewhat  protec- 
tive in  its  relation  to  the  host,  in  that  the  normal  fermentative  activities  of  the  organisms 
comprising  the  intestinal  flora  create  conditions  throughout  the  alimentary  canal  which 
are  inimical  to  the  development  of  alien  proteolytic  and  fermentative  bacteria. 

7  Centralbl.  f.  Bakt.,  1887,  ii,  633;   also,  Jahrb.  f.  Kinderheilk.,  1900,  lii,  1. 


590  GASTRO-INTESTINAL  BACTERIOLOGY 

flora  in  health  is  the  chemical  composition  of  the  ingested  food.1 
Escherich,2  as  far  back  as  1887,  clearly  showed  that  a  very  charac- 
teristic change  in  the  intestinal  flora  of  dogs  could  be  brought  about 
by  feeding  protein,  during  which  bacteria  that  liquefy  gelatin  become 
abundant  in  the  feces. 

Assuming  that  food  is  an  important  factor  in  determining  the  more 
common  types  of  bacteria  found  respectively  in  the  intestinal  tracts 
of  nurslings,  artificially  fed  children  and  adults,  it  would  be  reasonable 
to  expect  that  the  same  or  similar  bacteria  should  develop  in  the 
intestinal  tracts  of  experimental  animals,  provided  they  were  fed  upon 
the  same  foods  as  nurslings  or  adults.  A  prolonged  series  of  experi- 
ments upon  monkeys,3  dogs  and  cats  have  shown  that  alternations  in 
diet  do  influence  the  prevailing  types  of  bacteria  in  the  intestinal 
tract  to  a  marked  degree.  The  essential  features  of  these  experiments 
were  that  monkeys,  dogs  and  cats  fed  upon  cow's  milk  containing 
sufficient  lactose  solution  to  bring  the  percentage  of  protein  and 
carbohydrate  approximately  to  that  of  human  breast  milk  excreted 
feces  which,  in  appearance  and  in  bacterial  content,  approached  very 
closely  those  of  the  normal  human  nursling.  The  acid  reaction,  prac- 
tical absence  of  obligately  proteolytic  bacteria,  the  dominance  of 
Bacillus  bifidus  and  acidophilus  and  the  appearance  of  Micrococcus 
ovalis  in  numbers  similar  to  corresponding  types  in  normal  nurslings' 
feces  were  in  striking  contrast  to  the  feces  of  the  same  animal  after 
a  prolonged  feeding  with  a  purely  protein  diet.  In  the  latter  event 
large  numbers  of  proteolytic  bacteria  were  present  in  the  feces,  which 
were  alkaline  in  reaction  and  rich  in  indol,  phenols,  hydrogen  sulphide, 
ammonia  and  other  products  indicative  of  intense  proteolytic  decom- 
position. Obligately  fermentative  bacteria  of  the  bifidus-acidophilus 
type  were  few  in  number,  or  practically  absent.  Recently  Rettger4 
has  made  somewhat  parallel  observations  in  mice  and  rats.5 

The  nature  of  the  dominant  organisms  which  develop  in  diets  rich 
in  carbohydrate  varies  with  the  carbohydrate  itself.  Bacillus  bifidus 
is  more  commonly  predominant  when  lactose  is  the  sugar  fed,  without 

1  See  Kendall,  Jour.  Med.  Research,  1911,  xxv,  136,  for  resume. 

2  Darmbaktericn,  etc.,  p.  111. 

3  Kendall,  Jour.  Biol.  Chem.,  1909,  vi,  499.     Herter  and  Kendall,  ibid.,  1910,  vii,  203. 
Kendall,  Jour.  Med.  Research,  1910,  xxii,  153;    ibid.,  1911,  xxiv,  411;    1911,  xxv,  117. 

4  Centralbl.  f.  Bakt.,  prig.,  1914,  Ixxiii,  362. 

6  It  is  rather  more  difficult  to  replace  a  proteolytic  flora  in  adult  animals  by  a  fer- 
mentative flora  than  it  is  in  young  animals  of  the  same  species;  the  explanation  of  this 
relative  refractoriness  to  substitution  of  obligately  fermentative  types  of  bacteria  for 
the  facultative  organisms  commonly  found  in  the  intestinal  tracts  of  the  older  animal 
is  by  no  means  clear. 


THE  GASTRO-INTESTINAL  FLORA   OF  NORMAL  INFANTS    591 

an  excess  of  protein;  if  maltose  or  dextrose  is  substituted  for  lactose 
under  the  same  conditions,  Bacillus  acidophilus  is  very  frequently 
the  more  prominent.  In  like  manner,  the  nature  of  the  protein 
influences  the  types  of  proteolytic  bacteria  to  a  very  marked  degree; 
in  general,  animal  proteins  other  than  casein  appear  to  encourage  a 
somewhat  more  active  proteolytic  flora  than  vegetable  proteins.  These 
observations  are  in  harmony,  in  essential  features  at  least,  with  those 
made  under  like  conditions  in  man.  A  monotonous  diet  in  which  lac- 
tose and  protein  are  fed  in  proportions  and  amounts  similar  to  breast 
milk  leads  to  the  gradual  development  of  an  intestinal  flora  in  experi- 
mental animals  closely  simulating  that  of  nurslings.  A  preponderance 
of  protein,  on  the  other  hand,  encourages  the  development  of  bacteria 
which  are  more  proteolytic  in  nature. 

It  is  a  striking  fact  that  the  above  alternation  in  intestinal  bacteria 
following  changes  along  definite  lines  in  the  diet  is  eliciteo!  only  when 
the  feeding  is  maintained  for  several  days;  rapid  alternations  between 
a  purely  protein  diet  and  a  diet  rich  in  sugar  (as  cow's  milk  diluted 
with  an  equal  volume  of  4  per  cent,  lactose  solution)  do  not  ordinarily 
lead  to  such  noteworthy  changes  in  the  types  of  bacteria  excreted  in 
the  feces.1  The  general  trend  of  such  rapid  alternations  between  a 
protein  regimen  and  one  in  which  sugars  predominate  (starches  do 
not  necessarily  react  in  this  manner)  is  to  establish  a  flora  which  is 
relatively  heterogeneous,  in  which  there  is  neither  a  decided  predom- 
inance of  obligately  carbohydrophilic  bacteria,  as  B.  bifidus  or  acido- 
philus, nor  of  obligately  proteolytic  bacteria. 

A  most  striking  and  important  influence  of  diet  upon  bacterial 
activity  in  the  intestinal  tract  does  not  manifest  itself  in  a  study  con- 
fined exclusively  to  the  changes  in  bacterial  types  of  the  intestinal 
flora.  The  monotony  of  the  typical  nursling  flora  depends  in  a  large 
measure  on  the  continual  presence  of  lactose  (a  sugar  not  fermented 
by  a  majority  of  bacteria)  throughout  the  intestinal  tract.  A  sub- 
stitution of  other  sugars — as  dextrose,  saccharose  or  maltose — leads 
to  a  replacement  of  Bacillus  bifidus  by  other  more  or  less  obligately 
fermentative  organisms,  provided  an  excess  of  the  respective  carbo- 
hydrate be  maintained,  but  the  same  monotony  of  types  is  observed. 

The  proportion  of  carbohydrate  to  protein  in  the  diet  of  normal 
adults  is  far  less  than  in  nurslings  and,  furthermore,  a  considerable 
proportion  of  the  carbohydrate  is  in  the  form  of  starches  which,  as 

1  This  probably  explains  some  of  the  irregularities  experienced  during  brief  feeding 
experiments. 


592  G ASTRO-INTESTINAL  BACTERIOLOGY 

such,  are  not  readily  fermented  by  most  bacteria.  Again,  sugars,  if 
they  are  present,  are  largely  absorbed  from  the  higher  levels  of  the 
small  intestine,  leaving  residual  unhydrolyzed  starches  and  protein 
in  relatively  great  concentration  in  the  lower  levels  of  the  large  intes- 
tine. It  is  not  surprising,  under  these  conditions,  to  find  that  the 
more  obligate  fermentative  bacteria — the  Cocci — are  prominent  at 
the  higher  levels,  as  is  the  case  normally  in  infants;  that  facultative 
bacteria,  as  Bacillus  coli,  are  common  in  a  transitional  zone  between 
a  medium  containing  moderate  amounts  of  utilizable  carbohydrate 
and  one  in  which  the  utilizable  carbohydrate  is  frequently  absent,1 
and  finally,  that  proteolytic  organisms  are  most  abundant  in  the  large 
intestines,  where  carbohydrate  in  significant  amounts  is  practically 
absent,  but  where  the  protein  concentration  is  still  considerable. 
Practically  all  the  bacteria  found  in  the  large  intestine  of  normal 
adults  exhibit  a  preferential  action  upon  dextrose  (a  product  of  the 
hydrolysis  of  starches  and  many  bioses  as  well),  but  they  are,  for 
the  most  part,  unable  to  utilize  lactose. 

There  are,  therefore,  two  important  factors  to  consider  in  dis- 
cussing the  influence  of  diet  upon  the  intestinal  flora:  The  substitution 
of  types  of  organisms,  which  frequently  follows  a  monotonous  diet; 
and  a  change  in  the  metabolism  of  existing  types  of  intestinal  bacteria 
when  dietary  conditions  are  such  that  the  intestinal  medium  at  one 
or  another  level  fluctuates  in  its  content  of  utilizable  carbohydrate 
and  other  nutrient  substances.2 

From  time  to  time  modifications  or  changes  in  the  types  of  bacteria 
in  the  intestinal  flora  and  of  their  activities  takes  place.  The  nature 
and  extent  of  these  modifications  and  their  effects  upon  the  host  vary 
very  much,  not  only  qualitatively,  but  quantitatively  as  well.  An 
invasion  of  the  intestinal  tract  by  exogenous  bacteria,  as  the  dysen- 
tery bacillus  or  the  cholera  vibrio,  may  lead  to  a  more  or  less  pro- 
nounced replacement  of  some  of  the  normal  intestinal  types  by  these 
alien  organisms,  and  to  the  production  of  disease.  Normal  intestinal 
organisms  or  types  indistinguishable  from  them  by  ordinary  methods 
of  study  also  may  multiply  with  abnormal  luxuriance  through  unusual 

1  Bacillus  coli  and  various  closely  related  bacilli  are  among  the  most  labile  of  intes- 
tinal bacteria  in  adapting  their  metabolism  to  the  composition  of  the  intestinal  contents. 
In   a   medium   containing   both   utilizable   carbohydrate   and   utilizable   protein   these 
organisms  act  principally  upon  the  carbohydrate,  forming  lactic  and  smaller  amounts 
of  other  acids.     In  a  protein  medium  the  products  of  metabolism  are  indol,  phenols, 
and  other  products  of  proteolysis. 

2  For  a  brief  general  discussion  of  the  influence  of  nutritional  factors  upon  bacterial 
metabolism,  see  Section  on  Bacterial  Metabolism. 


THE  GASTROINTESTINAL  FLORA  OF  NORMAL  INFANTS      593 

conditions,  extend  their  normal  habitat,  and  crowd  out  some  of  the 
existing  organisms,  eventually  leading  to  abnormal  reactions  in  the 
alimentary  canal  which  may  be  detrimental  to  the  host. 

There  are  many  intestinal  disturbances  of  unknown  causation,  pre- 
sumably unrelated  to  bacterial  activity,  which  naturally  are  not  of 
interest  in  this  connection.  There  is  a  second  group  of  conditions  in 
which  bacteria  may  conceivably  play  a  secondary  part;  in  some 
instances  abnormal  physiological  conditions  in  the  alimentary  canal 
may  be  justly  regarded  as  the  antecedent  factors.  The  boundaries 
of  these  two  groups  are  poorly  circumscribed  and  they  merge  through 
imperceptible  or  poorly"  defined  limits  into  a  third  group  of  cases  in 
which  the  activities  of  endogenous  or  exogenous  bacteria  in  the  alimen- 
tary canal  may  be  the  causative  factor  in  morbid  processes  of  the 
gastro-intestinal  tract. 

For  convenience  of  discussion  this  last  group  may  be  divided  into 
three  types:  (a)  Those  cases  in  which  products  resulting  from  the 
action  of  bacteria  upon  proteins  or  their  derivatives  appear  to  be  the 
prominent  factors  in  the  production  of  the  morbid  process;  (6)  those 
cases  in  which  products  resulting  from  the  fermentation  of  carbohy- 
drates by  the  action  of  bacteria  are  the  prominent  substances  concerned 
in  the  morbid  process.  A  third  group,  practically  unstudied  at  the 
present  time,  would  include  those  cases  in  which  symbiotic  activities 
of  proteolytic  and  fermentative  bacteria  would  result  in  the  production 
of  substances  derived  both  from  proteins  and  from  carbohydrates.1 

The  action  of  bacteria  on  fats  is  little  understood  at  present  and 
no  statement  can  be  made  covering  this  type  of  abnormality.  It  is 
expressly  understood  that  products  of  the  nature  of  endotoxins  result- 
ing from  the  dissolution  of  bacteria  are  not  considered  in  this  connec- 
tion, which  relates  exclusively  to  a  discussion  of  the  activities  of  living 
organisms. 

The  symptomatology  induced  from  the  products  arising  from 
the  decomposition  of  proteins  or  protein  derivatives  by  the  action 
of  bacteria  in  the  intestinal  tract  depends  largely  upon  the  organism 
or  organisms  concerned;  it  varies  from  the  somewhat  insidious,  slowly 
progressing,  so-called  auto-intoxication,  in  which  a  marked  increase 

1  Thus,  in  occasional  severe  diarrheas  of  children  strains  of  Bacillus  coli  and  Bacillus 
mesentericus  are  occasionally  isolated,  which  grow  symbiotically  in  milk,  causing  a  deep- 
seated  change  both  in  the  protein  and  carbohydrate  content  of  the  medium.  The  result 
of  their  mutual  development  is  much  greater  than  the  sum  of  their  separate  activities. 
Ordinary  strains  of  these  organisms  frequently  do  not  exhibit  this  symbiotism.  It  is  by 
no  means  improbable  that  similar  symbiotic  activity  in  the  intestines,  if  unrestrained, 
may  lead  to  conditions  incompatible  with  the  well-being  of  the  host. 
38 


594  GASTRO-INTESTINAL  BACTERIOLOGY 

of  urinary  ethereal  sulphates  may  be  a  suggestive  index,  to  the  acute 
toxemias  characteristic  of  bacillary  dysentery,  typhoid,  paratyphoid 
or  cholera.  Of  course,  a  variety  of  other  bacteria  than  the  few  men- 
tioned specifically  may  be  concerned,  either  alone  or  in  symbiosis. 
Thus  streptococci  alone  and  streptococci  in  association  with  dysentery 
bacilli  may  be  justly  regarded  as  the  etiological  agents  in  their  respec- 
tive syndromes.  The  important  factor,  from  the  viewpoint  of  this 
discussion,  is  to  realize  that  the  formation  of  nitrogenous  products 
from  proteins  or  protein  derivatives  which  are  being  utilized  by 
various  types  of  intestinal  bacteria  for  energy  may  be  injurious  to  the 
host.  These  substances  are  of  unknown  composition  for  the  most 
part,  but  beyond  doubt  they  are  nitrogenous.  Some,  as  phenols, 
cresols,  or  indol  are  simple  in  structure  and  ordinarily  harmless,  or 
nearly  so,  although  long-continued  absorption  may  gradually  lead  to 
cumulative  effects.  Others,  as  beta  imidazoleethylamine  and  other 
primary  amines  formed  from  amino  acids  may  be  physiologically 
active.  The  unknown  poisons  of  the  meat  poisoning  group  and  those 
characteristic  of  the  various  bacteria  which  cause  acute  infections 
of  intestinal  origin  are  of  unknown  structure  and  complexity. 

The  other  prominent  type  of  abnormal  bacterial  activity  in  the 
alimentary  canal — the  fermentative  type — is  of  entirely  different 
origin;  the  essential  factor  is  either  a  decomposition  of  carbohydrates, 
with  the  formation  of  products  abnormal  for  the  intestine,  or  of  excess 
of  normal  fermentative  products.  The  abnormality  may  be  a  simple 
hyperacidity,  as,  for  example,  that  caused  by  an  overgrowth  of  aciduric 
bacteria  when  certain  sugars,  as  maltose,  fed  in  too  large  amounts, 
lead  to  an  overdevelopment  of  the  aciduric  bacteria;  or  it  may  be 
more  complex.  This  happens  frequently  when  there  is  an  overgrowth 
of  Bacillus  aerogenes  capsulatus,  or  of  members  of  the  Mucosus  Cap- 
sulatus  Group.  In  the  latter  event  the  exact  nature  of  the  irritative 
substance  is  as  yet  unknown,  but  it  is  in  all  probability  not  a  nitro- 
genous compound.  It  is  formed  from  carbohydrates,  which  contain 
no  nitrogen.  The  factors  leading  to  an  overgrowth  of  these  organisms 
in  the  intestinal  tract  appear  to  be  an  excess  of  carbohydrate  and  a 
lack  of  normal  lactic-acid-forming  bacteria.  It  is  a  significant  fact 
that  diarrheal  cases  associated  with  an  overgrowth  of  the  gas  bacillus 
even  of  several  years'  duration  do  not  exhibit  signs  or  symptoms  of 
toxemia  in  spite  of  the  protracted  illness. 

It  is  unfortunate  that  practically  none  of  the  bacteria  which  incite 
intestinal  disturbances  or  illness  produce  soluble  toxins  against  which 


THE  GASTROINTESTINAL  FLORA   OF  NORMAL  INFANTS     595 

antitoxins  can  be  prepared;  sera  likewise  have  been  unsatisfactory. 
There  is  little,  therefore,  that  can  be  accomplished  serologically 
with  present  methods  in  the  treatment  of  intestinal  disturbances  of 
bacterial  causation.  Attempts  to  permanently  eliminate  or  destroy 
undesirable  bacteria  with  cathartics  and  intestinal  antiseptics  have 
not  been  productive  of  results  in  the  past1  and  prolonged  starvation2 
per  se  does  not  lead  to  intestinal  sterility  or  to  a  significant  reduction 
in  the  offending  bacteria. 


X-' 


?$!& 


TvS* 


FIG  97. — Bacillus  bulgaricus.     (Photograph  by  Dr.  J.  H.  Stebbins,  Jr.,  from  the 
Fairchild  culture  of  the  Bacillus  bulgaricus. 

There  are  two  ways,  however,  in  which  direct  influence  may  be 
applied  to  bacteria  in  the  intestinal  tract :  By  a  substitution  of  harm- 
less types  of  organisms  for  abnormal  types,  and  by  varying  the  diet 
of  the  host  in  such  a  manner  that  the  intestinal  contents  at  the  desired 
level  shall  contain  nutritive  substances  that  may  be  reasonably 
expected  to  shift  the  metabolism  of  the  offending  organism,  and 
therefore  radically  change  the  character  of  the  products  of  its 
metabolism. 

A  substitution  of  bacteria  may  be  accomplished,  theoretically  at 
least,  either  by  feeding  cultures  of  organisms  whose  products  of  growth 

1  Kendall,  Jour.  Med.  Research,  1911,  xxv,  149,  for  brief  resume. 

2  Even  after  thirty-one  days'  starvation,  a  large  number  of  viable  bacteiia  were  found 
in  the  lower  part  of  the  intestinal  tract  of  the  one  case  studied  with  this  possibility  in  view. 


596  GASTRO-INTESTINAL  BACTERIOLOGY 

are  harmless  to  the  host  and  more  or  less  inimical  to  the  bacteria  it 
is  desirable  to  supplant,  or  by  administering  a  diet  which  contains 
appropriate  nutritive  substances  in  sufficient  amounts  to  create  con- 
ditions favoring  the  development  of  normal  intestinal  bacteria  whose 
activities  are  in  opposition  to  those  it  is  desired  to  restrict  or  supplant. 

The  effects  of  a  monotonous  diet  maintained  for  considerable  periods 
of  time  upon  the  intestinal  flora  of  a  normal  individual  are  clearly 
shown  in  the  normal  nursling,  where  intestinal  organisms  are  largely 
carbohydrophilic  and  fermentative  in  character.  Feeding  experiments 
in  normal  animals  indicate_that  the  development  of  a  nursling  intes- 
tinal flora  follows  the  prolonged  administration  of  a  nursling  diet. 

If  the  intestinal  flora  to  be  modified  does  not  contain  sufficient 
numbers  of  the  desired  types  of  bacteria,  or  if  these  latter  organisms 
are  inactive,  it  may  be  important  to  reenforce  the  weakened  or  inactive 
residual  types  with  suitable  cultures  from  without.  Herter1  was  the 
first  to  recognize  the  possibility  of  introducing  desirable  types  of  bac- 
teria into  the  alimentary  canal  and  Metchnikoff2  has  extended  and 
popularized  this  form  of  bacteriotherapy  through  his  extensive  studies 
upon  the  effects  of  milk  soured  with  the  Bulgarian  bacillus  as  a 
therapeutic  measure  in  excessive  intestinal  putrefaction.  The  Bul- 
garian bacillus3  is  a  large  Gram-positive  organism,  which  is  non- 
motile  and  forms  neither  spores  nor  capsules.  It  develops  feebly  in 
ordinary  media,  but  luxuriantly  in  milk,  producing  considerable 
amounts  of  lactic  and  other  acids,  but  no  gas.  It  is  a  milk  parasite, 
having  been  perpetuated  in  this  medium  for  many  decades  by  the 
Bulgarian  peasants. 

The  underlying  principles  of  sour  milk  therapy  as  set  forth  by  Metch- 
nikoff. are:  a  restriction  of  the  protein  in  the  diet,  to  reduce  the 
available  putrescible  material  in  the  intestinal  tract;  and  the  adminis- 
tration of  liberal  amounts  of  sour  milk  to  flood  the  alimentary  canal 
with  preformed  lactic  acid.  It  was  originally  believed  that  the  Bul- 
garian bacillus  would  become  acclimatized  in  the  intestinal  tract  and 
continue  to  produce  lactic  acid  from  the  ingested  carbohydrate,  thus 
maintaining  an  acidity  throughout  the  intestinal  contents;  this  should 
create  conditions  inimical  to  the  development  of  putrefactive  organ- 
isms, which  are  said  to  be  intolerant  of  acids.  It  is  doubtful  if  the 
Bulgarian  bacillus  does  become  acclimatized  in  the  large  intestines, 

1  British  Med.  Jour.,  1897,  ii,  1847. 

2  Prolongation  of  Life. 

3  See  Rahe,  Jour.  Inf.  Dis.,  1914,  xv,  141,  for  description  and  differentiation  from 
other  aciduric  bacteria. 


THE  GASTRO-INTESTINAL  FLORA  OF  NORMAL  INFANTS,     597 

where  putrefactive  action  is  maximal.1  The  theoretical  and  practical 
difficulties  of  acclimatizing-  a  milk  parasite  in  the  intestinal  tract 
would  suggest  that  a  normal  intestinal  organism  of  the  lactic-acid 
type,  as  Bacillus  acidophilus2  (whose  habitat  is  the  large  intestine), 
would  be  theoretically  more  efficient  in  those  cases  where  Bacterio- 
therapy  is  indicated. 

Bromatherapy. — The  very  direct  and  striking  relation  between 
the  nature  of  the  food  of  bacteria  and  the  character  of  their  products 
of  metabolism  has  an  important  theoretical  and  practical  application 
in  relation  to  intestinal  bacteriology  in  health  and  disease.  It  has 
been  stated  in  another  section  that  products  of  bacterial  metabolism 
harmful  to  the  host  may  be  classified  as  nitrogenous  compounds 
derived  from  proteins  and  protein  derivatives,  and  non-nitrogenous 
compounds  derived  from  carbohydrates  and  fats.  The  former  are 
produced  by  bacteria  acting  upon  proteins  and  their  derivatives  in 
the  absence  of  utilizable  carbohydrates;  the  latter  are  formed  by 
bacteria  which  are  utilizing  carbohydrates  or  fats.  Thus,  the  diph- 
theria bacillus  forms  a  powerful  toxin  in  protein  media,  but  does  not 
form  toxin  when  available  carbohydrate  is  added  to  the  medium; 
Bacillus  coli  forms  indol  in  protein  media,  but  does  not  form  indol 
when  available  carbohydrate  is  added  to  the  medium.  If  these  bac- 
teria were  developing  in  the  intestinal  tract  at  levels  where  a  contin- 
uous supply  of  caibohydrate  could  reach  them  it  would  be  theoretically 
possible  to  reduce  or  even  prevent  the  formation  of  toxin  or  indol 
respectively  when  utilizable  carbohydrates  are  present. 

There  are  a  number  of  intestinal  conditions  of  bacterial  causation 
in  which  available  evidence  points  strongly  to  the  formation  of  pro- 
ducts arising  from  the  metabolism  of  protein  or  protein  derivatives 
by  specific  organisms  as  important  etiological  factors  in  the  morbid 
process.  Thus  cholera,  bacillary  dysentery,  typhoid,  paratyphoid  and 
many  less  acute  infections  are  associated  definitely  with  the  develop- 
ment of  these  organisms  within  the  body  and,  to  some  degree  at  least, 
at  the  expense  of  the  body  tissues. 

All  of  these  organisms  produce  lactic  and  other  acids  when  suitable 
carbohydrates  are  available;  the  products  of  fermentation  of  these 
bacteria,  chiefly  lactic  and  other  acids,  are  almost  certainly  no  more 
harmful  to  the  host  than  are  those  formed  by  Bacillus  bulgaricus, 

1  Herter  and  Kendall,  Jour.  Biol.  Chem.,  1908,  v,  293;    Rahe,  Jour.,  Inf.  Dis.,  1915, 
xvi,  210. 

2  Rotch  and  Kendall,  Am.  Jour.  Dis.  of  Children,  1911,  ii,  30. 


598  GASTRO-INTESTINAL  BACTERIOLOGY 

Bacillus  coli  or  Bacillus  acidophilus,  produced  under  like  conditions. 
In  other  words,  available  evidence  points  strongly  to  the  view  that 
cholera  vibrios,  typhoid,  dysentery  and  paratyphoid  bacilli  and  similar 
organisms  produce  their  characteristic  and  harmful  effects  when  they 
are  developing  in  media  free  from  utilizable  carbohydrate;  when 
utilizable  carbohydrates  are  added  to  these  media,  non-characteristic, 
harmless  products  are  formed.  It  is  frankly  admitted  that  the  chemis- 
try of  the  products  of  nitrogenous  metabolism  of  pathogenic  bacteria 
is  wholly  unknown,  and  a  rigorous  proof  of  a  relation  between  nitro- 
genous metabolism  and  disease  is  yet  to  be  elucidated;  the  significant 
fact  that  the  products  of  fermentation  of  these  organisms  are  almost 
certainly  innocuous  to  the  host  cannot  be  disregarded. 

In  the  absence  of  any  definite  indication  to  the  contrary  it  would  be 
logical  to  attempt,  to  maintain  a  sufficient  concentration  of  carbo- 
hydrate within  the  intestinal  canal  in  these  infections  as  a  therapeutic 
measure.  This  would  be  advantageous  to  the  patient  as  a  physio- 
logical procedure,  as  Coleman  and  Shaffer1  have  shown  in  their  clas- 
sical studies  in  typhoid,  and  it  would  provide  continuously  at  least 
a  minimal  amount  of  readily  utilizable  carbohydrate  which  would 
shift  the  metabolism  of  all  the  intestinal  organisms,  pathogenic  and 
non-pathogenic,  in  such  a  manner  that  harmless  lactic  acid  would  be 
formed  by  them.  The  bacteria  under  these  conditions  would  theoreti- 
cally, and  in  all  probability  practically,  derive  their  energy  from  the 
readily  fermentable  carbohydrate  and  thus  not  only  minimize  their 
action  upon  the  proteins  of  the  intestinal  contents,2  but  would  tend  to 
create  an  acid  reaction  there  which  in  itself  would  be  a  potent  agent  in 
restricting  the  activity  of  the  pathogenic  organisms  in  the  alimentary 
tract. 

The  associated  bacteria  of  the  intestinal  tract  also  form  acids  under 
these  conditions;  Bacillus  coli  does  not  form  indol,  and  other  products 
of  putrefaction  are  absent.  Within  a  few  days,  under  favorable  cir- 
cumstances, the  cumulative  effect  of  a  diet  liberal  in  carbohydrate 
will  lead  to  a  considerable  development  of  aciduric  bacteria,  especially 

1  Arch.  Int.  Med.,  1909,  iv,  538. 

2  It  is  a  well-attested  fact  that  typhoid  bacilli  develop  within  the  tissues  of  the  body, 
and  it  might  appear  that  a  carbohydrate  diet  would  therefore  be  ineffective ;  it  is  import- 
ant to  remember  that  the  blood  normally  contains  about  0.08  per  cent,  dextrose,  an 
amount  amply  sufficient  to  protect  protein  from  their  attack.     A  liberal  carbohydrate 
diet  should  tend  to  maintain  the  concentration  of  blood  sugar  at  its  physiological  level. 
Recently  Simonds  (Jour.  Inf.  Dis.,  1915)  has  shown  that  the  products  arising  from  the 
autolysis  of  typhoid  bacilli  grown  in  dextrose  media  are  decidedly  less  toxic  for  rabbits 
than  those  grown  in  dextrose-free  media  when  acted  upon  by  specific  lytic  sera.     This 
observation  may  well  have  an  important  bearing  upon  the  case  in  question. 


THE  GASTRO-INTESTINAL  FLORA  OF  NORMAL  INFANTS     599 

of  the  bifidus-acidophilus  type  if  any  be  present  in  the  alimentary 
canal  to  start  with.1  The .intestinal  contents  are  acid  in  reaction  at 
this  time  and  unfavorable  for  the  development  of  the  pathogenic 
types. 

It  must  be  realized  that  a  number  of  conditions  may  reduce  the 
theoretical  efficiency  of  a  diet  rich  in  carbohydrate  in  intestinal  infec- 
tions; not  infrequently  the  intestinal  mucosa  is  inflamed  and  covered 
with  an  exudate  of  mucus  and  serum,  alkaline  in  reaction  and  rather 
impermeable  to  intestinal  medication.  Stasis  in  the  large  intestine 
will  frequently  lead  to  a  residue  of  protein  derivatives  there,  quite 
free  from  carbohydrate,  because  the  latter  is  readily  hydrolyzed  and 
absorbed  as  dextrose.  There  may  be,  and  undoubtedly  is,  in  some 
cases,  a  deficiency  of  the  more  effective  lactic-acid-forming  bacteria 
in  the  intestinal  contents;  whatever  organisms  are  present,  however, 
almost  without  exception  form  acids  from  carbohydrate,  especially 
dextrose.  The  possibility  of  an  overgrowth  with  the  gas  bacillus  must 
be  borne  in  mind  if  considerable  quantities  of  sugars  are  to  be 
administered. 

Notwithstanding  these  difficulties,  a  diet  rich  in  carbohydrate  has 
been  shown  to  be  well  tolerated  in  this  type  of  infection,  be  it  acute  or 
chronic.  Coleman  and  Shaffer,2  using  the  high  calory  diet  of  the 
former  in  typhoid  fever,  have  shown  by  careful  chemical  studies  that 
the  severe  loss  of  nitrogen  and  of  weight  which  occurs  on  a  low  calory 
diet  can  be  very  largely  prevented  by  a  diet  comparatively  rich  in 
carbohydrate,  and  the  symptoms  of  toxemia  are  materially  reduced 
as  well.  Torrey3  has  shown  that  the  changes  in  the  intestinal  flora 
in  typhoid  fever  with  the  Coleman  diet  are,  in  general,  a  replacement 
of  the  more  proteolytic  bacteria  by  greater  or  lesser  numbers  of 
aciduric  organisms,  a  change  similar  to  that  observed  in  bacillary 
dysentery,4  in  which  the  same  general  plan  of  liberal  feeding  of  lactose 
was  tried.  The  reduction  in  symptoms  of  toxemia  in  typhoid  patients 
following  a  high  calory  diet  including  several  ounces  of  lactose  is 
significant;  it  can  hardly  be  explained  entirely  on  the  theory  of  cal- 
ories; it  is  very  probable  that  a  change  in  the  metabolism  of  the 
typhoid  bacillus  is  a  potent  factor  in  this  phenomenon. 

1  Kendall,  Boston    Med.  and    Surg.  Jour.,  1910,  clxiii,  398;    1911,  clxiv,  288;  Jour. 
Am.  Med.  Assn.,  1911,  Ivi,  1084;  Jour.  Med.  Research,  1911,  xxiv,  411;    1911,  xxv,  117. 
Kendall  and  Walker,  Boston  Med.  and  Surg.  Jour.,   1911,  clxiv,  301.     Kendall  and 
Smith,  ibid.,  1911,  clxv,  306.     Kendall,  Bagg  and  Day,  ibid.,  1913,  clxix,  741.     Kendall 
and  Day,  ibid.,  1913,  clxix,  753. 

2  Arch.  Int.  Med.,  1909,  iv,  538. 

3  Jour.  Inf.  Dis.,  1915,  xvi,  72.       4  Kendall,  Boston  Med.  and  Surg.  Jour.,  1911,  clxiv. 


600  G ASTRO-INTESTINAL  BACTERIOLOGY 

To  summarize,  the  important  effects  to  be  accomplished  by  a  liberal 
carbohydrate  diet  in  those  infections  where  the  decomposition  of 
proteins  or  protein  derivatives  by  bacterial  activity  leads  to  chronic 
or  acute  illness  of  intestinal  origin  are — a  change  in  the  metabolism 
of  the  offending  organism  resulting  in  the  formation  of  lactic  and 
other  acids  in  them  in  place  of  putrefactive  products,  and  a  gradual 
replacement  of  the  proteolytic  and  pathogenic  types  by  bacteria  of 
the  fermentative  varieties. 

Another  type  of  intestinal  disturbance  depends  upon  an  unusual 
or  an  excessive  decomposition  of  carbohydrate.  The  excessive  forma- 
tion of  acid  within  the  intestinal  tract  by  an  overgrowth  of  aciduric 
bacteria  is  well  illustrated  in  young  infants,  especially  those  fed  upon 
too  much  maltose.1  The  dietary  treatment  of  such  cases  is  too  obvious 
to  require  further  remarks.  A  group  of  cases  which  vary  in  severity 
from  mild,  long-continued  diarrhea  of  several  years'  duration  to  very 
severe  acute  bloody  diarrhea  with  great  prostration  are  apparently 
caused  by  an  overgrowth  of  the  gas  bacillus  in  the  intestinal  tract. 
This  organism. is  relatively  intolerant  of  lactic  acid,  and  a  diet  prac- 
tically free  from  carbohydrate,  rich  in  protein,  and  reenforced  by 
a  liberal  consumption  of  very  acid  buttermilk  usually  effects  a  rapid 
improvement  in  the  acute  cases,  and  a  gradual  improvement  in  those 
cases  which  are  of  months'  or  years'  duration.  .Members  of  the  Mucosus 
Capsulatus  Group  of  bacteria  may  also,  by  overgrowth,  set  up  a  fer- 
mentative type  of  diarrhea  which  resembles  that  of  the  gas  bacillus 
in  its  general  features.  The  dietary  treatment  of  these  cases  is  like 
that  of  gas  bacillus  diarrheas. 

1  Kendall,  Boston  Med.  and  Surg.  Jour.,  1910,  cixiii,  322. 


SECTION  V. 

APPLIED  BACTERIOLOGY. 

CHAPTER  XXXI. 
BACTERIOLOGY  OF  MILK. 

A  VERITABLE  river  of  milk,  collected  from  many  sources,  flows 
daily  into  the  larger  cities  of  the  country.  Milk  is  an  important  food, 
particularly  for  infants  and  children,  partly  because  it  is  relatively 
inexpensive  and  requires  little  or  no  preliminary  preparation,  chiefly 
because  it  contains  in  a  small  volume,  all  the  essential  nutritive 
elements  combined  in  readily  utilizable  form.  Herein  lies  its  potential 
danger.  It  is  a  good  culture  medium  for  bacteria  and  its  opacity  pre- 
cludes the  possibility  of  visually  detecting  the  contamination.  Indeed, 
considerable  amounts  of  dirt  and  filth  may  be  introduced  into  milk 
without  visibly  changing  its  normal  appearance. 

It  is  inevitable,  from  existing  conditions,  that  milk  from  many 
sources  must  be  mixed  before  it  appears  in  the  open  market;  there 
may  be  an  element  of  danger  or  a  measure  of  safety  in  this  homo- 
genizing process.  If  milk  from  a  single  dairy  happens  to  be  infected 
with  pathogenic  bacteria,  the  degree  of  infection  may  be  sufficient  to 
effectively  seed  the  entire  volume  with  which  it  is  mingled,  or  the 
degree  of  dilution  may  reduce  the  numbers  of  bacteria  per  volume 
below  the  danger  point  of  infection  for  man. 

The  various  manipulations  to  which  milk  is  necessarily  subjected 
before  it  reaches  the  consumer  afford  ample  opportunity  for  bacterial 
contamination  and  the  time  which  necessarily  elapses  between  pro- 
duction and  consumption  furnishes  one  of  the  additional  elements 
necessary  for  the  development  of  adventitious  bacteria.  The  tem- 
perature at  which  the  milk  is  maintained  is  another  important  physical 
element  which  determines  the  extent  of  bacterial  growth  in  it. 

A  moderate  number  of  bacteria  pathogenic  for  man  may  lead  to 
infection  of  those  who  drink  milk  containing  them,  even  if  no  develop- 
ment of  these  organisms  has  taken  place.  On  the  other  hand,  the 


602  BACTERIOLOGY  OF  MILK 

growth  of  bacteria  ordinarily  not  regarded  as  pathogenic  may  induce 
changes  in  this  medium  which  render  it  unfit  or  even  harmful  for 
human  use.  If  these  changes  are  not  of  sufficient  magnitude  to  alter 
the  physical  appearance  of  the  milk,  or  if  they  are  not  perceptible  to 
the  senses,  they  may  easily  escape  detection  and  yet  lead  to  illness 
of  the  consumer.  It  is  obvious,  therefore,  that  those  very  elements 
which  make  milk  a  valuable  food  create  conditions,  themselves  innocu- 
ous, through  which  it  may  become  actively  or  passively  a  vehicle  for 
the  transmission  of  disease  to  man. 

One  of  the  great  hygienic  problems  of  the  present  time  is  that  of 
maintaining  and  safeguarding  the  milk  supply. 

Sources  of  Bacterial  Contamination  of  Milk. — Milk  freshly  drawn 
from  the  udder  of  a  healthy  cow,  although  practically  never  sterile, 
rarely  contains  many  bacteria.  The  greatest  contamination  of  milk 
probably  takes  place  from  unsterile  utensils,  although  undoubtedly 
unclean  animals,  filthy  surroundings  and  dusty  air  contribute  many 
bacteria  to  it.  Organisms  introduced  into  milk  from  the  hands  of  the 
milker  and  from  his  respiratory  tract  may  be  far  more  formidable  to 
the  consumer  than  mere  numbers  of  saprophytic  bacteria. 

The  ever-increasing  application  of  complicated  machinery  for 
handling  and  bottling  milk,  while  reducing  to  a  large  degree  the  possi- 
bility of  contamination  from  human  sources,  provides  a  fruitful  source 
of  contamination  with  saprophytic  organisms.  The  sterilization  of 
machinery  of  this  type  is  difficult  to  accomplish  and  not  infrequently 
incomplete  cleansing  between  periods  of  actual  use  leaves  a  residuum 
of  fluid  sufficiently  rich  in  nutritive  substances  to  permit  of  extensive 
bacterial  development.  The  first  portion  of  milk  run  through  a 
machine  in  this  condition  must  inevitably  be  grossly  seeded  with 
microorganisms. 

The  development  of  bacteria  which  have  gained  entrance  to  milk 
depends  to  a  very  considerable  degree  upon  the  temperature  at  which 
the  milk  is  kept  and  the  time  which  elapses  between  production  and 
consumption. 

Estimation  of  the  Bacterial  Content  of  Milk. — It  is  obvious  from 
the  preceding  observations  that  adventitious  milk  bacteria  may  be 
harmful  to  man  either  because  they  are  pathogenic  or  because  they 
produce  changes  in  the  composition  of  milk  which  make  it  unfit  for 
human  consumption.  From  an  hygienic  point  of  view,  therefore, 
milk  offered  for  sale  should  be  free  from  pathogenic  microorganisms 
and  of  low  bacterial  content. 


ESTIMATION  OF  THE  BACTERIAL  CONTENT  OF  MILK      603 

The  numbers  of  bacteria  in  milk  are  determined  in  practice  by  two 
distinct  methods : 

(a)  The   numbers   of   organisms   which   will   grow   upon   ordinary 
laboratory  media,  as  nutrient  agar  (cultural  count),  and: 

(b)  By  direct  microscopic  count. 

(a)  Cultural    Count.     Method:    1    c.c.  of    a    well-mixed    sample 
of    milk  is  diluted  ^Q,   1^5,  loibo  or   even  IOOOGO  with   sterile  water, 
depending  upon  the  grade  of  the  sample,  and  plated  on  nutrient 
agar.    The  number  of  colonies  which  develop  after  forty-eight  hours' 
incubation  at  37°  C.  multiplied  by  the  dilution  is  taken  as  the  bac- 
terial count  of  the  milk.    It  is  customary  in  some  laboratories  to  make 
a  parallel  count  at  20°  C.,  after  four  days'  incubation.    The  numbers 
of  colonies  developing  on  agar  at  the  lower  temperature  may  be  much 
greater  than  those  incubated  at  body  "temperature.     The  difference 
between  the  counts  is  usually  more  marked  in  samples  of  milk  which 
have  been  maintained  for  some  time  at  a  relatively  low  temperature, 
and  in  ice-cream.    In  such  cases  bacteria  whose  minimal  temperature 
of  growth  is  relatively  low — 4°  to  12°  C. — may  multiply  with  con- 
siderable rapidity.     These  organisms  frequently  fail  to  develop  at 
37°  C. 

The  cultural  count  possesses  advantages  and  disadvantages.  The 
principal  advantages  are:  the  simplicity  of  the  method,  comparative 
accuracy  of  results  provided  uniform  conditions  are  maintained,  and 
some  differentiation  of  the  types  of  organisms  present  in  the  milk. 
The  disadvantages  are:  the  time  required  to  obtain  results — milk 
is  perishable  and  cannot  be  held  pending  examination  by  this  proce- 
dure. Furthermore,  by  no  means  all  the  bacteria  which  may  theoreti- 
cally gain  access  to  the  milk  will  grow  upon  plain  agar;  this  is  par- 
ticularly true  of  pathogenic  microorganisms.  Bacteria  which  remain 
adherent  in  groups  or  chains  are  frequently  not  separated  during  the 
shaking  of  the  sample  and  a  single  colony  may  originate  from  such 
a  clump  or  chain.  This  naturally  introduces  an  error  which  may  be 
very  considerable  if,  for  example,  a  long  chain  of  streptococci  develops 
as  a  single  colony. 

(b)  Direct  microscopic  count.    Milk  hygienists  have  long  recognized 
the  advantages  of  a  direct  estimation  of  the  bacterial  count  of  milk 
and  numerous  methods  have  been  proposed,  from  time  to  time,  to 
accomplish  this  object.    The  most  practical  method  thus  far  prescribed 
appears  to  be  that  of  Prescott  and  Breed.1    The  theory  involved  is  to 

1  Centralb.  f.  Bakt.,  1911,  1,  246. 


604  BACTERIOLOGY  OF  MILK 

spread  a  definite  volume  of  milk  upon  a  definite  area  on  a  glass  slide, 
evaporate  the  fluid,  fix  the  sediment  (which  contains  all  the  bacteria 
in  the  sample),  and  stain  it  in  such  a  manner  that  the  microorganisms 
are  distinctly  colored.  The  organisms  of  a  definite  area  are  counted 
under  the  microscope.  The  number  in  the  original  sample  are  readily 
computed,  knowing  the  volume  of  milk  examined,  the  area  over  which 
it  is  spread  and  the  size  of  the  microscopic  field. 

In  practice  0.01  c.c.  of  a  well-mixed  sample  of  milk  is  spread 
uniformly  over  an  area  of  1  square  centimeter  on  a  glass  slide.  (This 
area  is  readily  outlined  with  a  wax  pencil,  using  a  pattern  previously 
ruled  on  a  piece  of  paper  as  a  guide  and  following  the  outline  on  the 
glass  slide;  the  wax  pencil  mark  tends  to  limit  the  spread  of  the  milk 
beyond  the  limits  of  the  square.)  The  film  of  milk  is  then  air-dried 
or  dried  at  40°  C.,  immersed  in  absolute  methyl  alcohol  for  a  few  min- 
utes to  fix  the  sediment  to  the  slide  and  to  remove  some  of  the  milk 
lipoids  and  fats  which  interfere  somewhat  with  the  staining,  and 
stained  (after  drying),  with  aqueous  methylene  blue.  Alkaline 
methylene  blue  should  not  be  used  because  the  alkali  tends  to  loosen 
the  film  of  casein. 

The  bacteria  are  counted  with  an  oil  immersion  lens.  It  is  necessary 
to  adjust  the  optical  combination  of  lens  and  eye-piece  so  that  the 
diameter  of  the  microscopic  field  is  exactly  0.0016  cm.,  corresponding 
to  an  area  of  0.005  sq.  cm.  This  can  be  readily  accomplished  with  a 
stage  micrometer. 

Each  organism  in  a  microscopic  field  corresponds  to  one-five-hun- 
dred-thousandth the  number  in  a  cubic  centimeter  of  the  original 
sample  of  milk  (.005  X  0.01  =  0.00005),  because  0.01  c.c.  of  milk  was 
spread  on  an  area  of  1  sq.  cm.  and  ^  of  the  volume  is  viewed  in  the 
microscopic  field.  In  other  words,  the  microscopic  field  contains  the 
bacteria  of  500000  c.c.  of  the  original  sample  of  milk  and  it  is  potentially 
equivalent  to  an  agar  plate  culture  of  the  milk  in  a  dilution  of  500000. 

If  the  bacteria  were  uniformly  distributed,  the  number  of  bacteria 
observed  in  one  field  multiplied  by  500,000  would  give  directly  the 
number  of  bacteria  per  cubic  centimeter  in  the  milk;  usually,  however, 
the  organisms  are  somewhat  irregularly  distributed  and  in  practice 
several  fields  are  counted  and  the  average  number  of  organisms  per 
field  is  multiplied  by  500,000.  Duplicate  determinations  should  always 
be  made.  The  results  obtained  are  fairly  uniform  when  the  exact 
details  of  the  method  are  closely  followed. 

The  advantage  of  the  direct  microscopical  count  are:  a  very  material 


IDENTIFICATION  OF  BACTERIA  IN  MILK  605 

reduction  in  the  time  necessary  to  obtain  results;  milk  which  con- 
forms to  the  standard  may  be  quickly  passed.  Badly  contaminated 
milk  can  be  detected  by  simple  inspection  without  even  the  formality 
of  a  count.  There  are  also  certain  disadvantages.  All  bacteria  which 
are  stainable  with  methylene  blue  are  visible  by  this  method  and 
dead  organisms  as  well  as  those  which  are  viable  appear  in  the  count. 
This  is  a  decided  source  of  error  in  pasteurized  milk,  where  a  relatively 
large  proportion  of  bacteria  are  killed  by  heat;  the  method  also  does 
not  distinguish  sharply  between  different  types  of  organisms. 

On  the  whole,  the  advantages  very  materially  outweigh  the  disad- 
vantages and  employed  judiciously  the  method  is  of  great  practical 
value  in  the  bacterial  control  of  dairies  and  milk  supplies. 

The  information  obtained  by  the  bacterial  count  is  of  importance 
chiefly  from  the  viewpoint  of  the  past  history  of  the  milk.  Milk  pro- 
duced in  cleanly  surroundings,  handled  carefully  in  sterile  utensils, 
kept  cool  and  delivered  promptly,  should  contain  relatively  few 
bacteria.  If  the  milk  is  handled  properly  but  not  kept  cool  the 
numbers  of  organisms  usually  increase  greatly,  but  as  a  rule  the 
variety  of  organisms  present  will  be  limited.  Improperly  handled 
milk  kept  cool  will  frequently  exhibit  several  types  of  bacteria,  but 
not  necessarily  a  high  total  count.  A  consistent  low  count  with  but 
few  types  of  bacteria  usually  indicates  a  satisfactory  milk  supply. 

Identification  of  Bacteria  in  Milk. — The  bacterial  types  found. in 
milk  may  be  very  varied;  the  opportunity  for  contamination  does 
not  cease  when  the  milk  is  drawn  from  the  cow — every  step  in  the 
handling  of  the  milk  from  the  producer  to  the  consumer  offers  new 
avenues  for  infection.  A  catalogue  of  all  the  bacteria  which  have 
been  isolated  from  milk  would  be  very  extensive,  but  of  little  practical 
value.  Of  vastly  greater  importance  is  the  recognition  of  the  patho- 
genic organisms  which  may  be  transmitted  to  man  and  the  chemical 
changes  wrhich  ordinary  saprophytic  milk-bacteria  induce  in  it.  There 
are  relatively  few  bacteria  which  are  pathogenic  both  for  the  cow  and 
for  man.  Of  these,  the  bovine  tubercle  bacillus,  the  unknown  virus 
of  foot  and  mouth  disease  and  the  virus  of  the  disease  known  as  trem- 
bles of  cattle  are  transmissible  to  man,  the  latter  causing  a  well-defined 
symptom  complex  known  as  milk  sickness.  Goats,  particularly 
Maltese  goats,  infected  with  the  specific  organism  Micrococcus  meli- 
tensis,  transmit  the  disease  Malta  fever  to  man  through  their  milk.1 

1  The  detection  of  tubercle  bacilli  in  milk  has  been  discussed  in  the  chapters  on  tuber- 
culosis and  bacillus  abortus.  Malta  fever  has  been  discussed  in  the  chapter  on  Micro- 
coccus  melitensis  and  foot  and  mouth  disease  in  the  section  relating  to  filterable  viruses. 


606  BACTERIOLOGY  OF  MILK 

In  addition,  the  viruses  of  certain  infections  specific  for  man  may 
be  transmitted  in  milk.  These  organisms  gain  entrance  to  the  milk 
directly  from  human  sources,  incidental  to  the  various  handlings 
which  it  undergoes,  and  they  may  persist  in  unheated  milk  in  suffi- 
cient numbers  to  infect  the  consumers.  Typhoid,  diphtheria,  scarlet- 
fever,  epidemic  sore  throat  and  pseudodiphtheria  infection,  dysentery 
(bacillary),  various  types  of  epidemic  diarrhea  and  even  Asiatic 
cholera  are  the  more  important  diseases  thus  transmitted. 

Except  in  very  rare  instances,  specific  pathogenic  bacteria  other 
than  the  bovine  tubercle  bacillus  and  Micrococcus  melitensis.  have 
not  been  isolated  directly  from  milk.  The  evidence  of  the  transmis- 
sion of  pathogenic  bacteria  through  infected  milk  rests  largely  upon 
statistical  data.  It  is  very  conclusive,  however,  and  many  severe 
epidemics  of  typhoid  fever  and  other  infections  have  been  satisfac- 
torily traced  to  carriers  or  mild  cases  of  the  same  disease  among  those 
who  have  undoubtedly  handled  the  milk. 

Conradi,1  however,  appears  to  have  isolated  the  typhoid  bacillus  from 
infected  milk  which  was  shown  to  be  responsible  for  a  small  outbreak 
of  typhoid  fever,  and  Bruck2  and  others  have  shown  that  typhoid 
bacilli  and  similar  pathogenic  bacteria  may  persist  and  even  multiply 
in  the  presence  of  the  various  microorganisms  commonly  present  in 
ordinarily  good  grades  of  milk. 

The  virus  of  foot  and  mouth  disease  and  the  bovine  tubercle  bacillus 
have  been  detected  in  butter  and  cheese  prepared  from  milk  containing 
these  viruses. 

The  origin  and  relation  of  streptococci  to  milk-borne  epidemics  of 
septic  sore  throat  and  tonsillitis  have  been  subjects  of  controversy. 
There  appear  to  be  two  theories:  one  theory  maintains  that  the 
streptococci  are  of  bovine  origin  and  presumably  derived  from  the 
udders  of  cows  which  are  suffering  from  mastitis  or  garget.  The 
other  theory  assumes  that  these  streptococci  are  usually  of  human 
origin  and  have  gained  entrance  to  the  milk  at  some  stage  of  its  post- 
bovine  history.  Theobald  Smith3  and  Brown  have  made  an  extensive 
study  of  this  subject  and  their  conclusions  are  of  particular  interest 
in  this  connection.  They  state  that  "there  is  at  present  no  satis- 
factory evidence  that  bovine  streptococci  associated  with  mastitis 
or  garget  are  the  agent  of  tonsillitis  in  man.  Whenever  cases  of 

1  Centralbl.  f.  Bakt.,  Orig.,  1906,  xl,  31. 

2  Deutsch.  med.  Wchnschr.,  1903,  xxix,  460. 

3  Jour.  Med.  Research,  1911,  xxxi,  501. 


IDENTIFICATION  OF  BACTERIA  IN  MILK  607 

garget  are  suspected  as  sources  of  infection  in  man,  both  human  and 
bovine  types  should  be  looked  for." 

The  most  numerous  of  the  saprophytic  bacteria  commonly  found 
in  raw  milk  belong  to  the  group  of  organisms  which  form  lactic  acid, 
but  no  gas,  from  lactose.  They  are  frequently  referred  to  as  lactic 
acid  bacteria,  but  this  name  is  not  wholly  appropriate  nor  is  it  dis- 
tinctive; many  unlike  organisms  possess  this  property  in  common. 
The  best  known  and  most  widely  distributed  of  these  lactic  acid 
bacteria  is  a  streptococcus,  Streptococcus  lacticus,1  an  organism  which 
is  present  not  only  in  moderate  numbers  in  the  feces  of  the  cow,  but 
also  upon  the  udder  and  flanks  of  the  animal  as  well  if  cleanliness  is 
not  strictly  observed.  The  initial  infection  of  milk  with  Streptococcus 
lacticus  is  usually  not  extensive,  but  milk  appears  to  be  a  particularly 
favorable  medium  for  its  development  and  even  after  a  few  hours 
the  organism  may  have  increased  greatly  in  numbers  if  the  tempera- 
ture conditions  are  favorable.  The  most  noteworthy  chemical  change 
associated  with  the  growth  of  Streptococcus  lacticus  is  a  rapid  accu- 
mulation of  acid,  principally  lactic  acid,  which  soon  results  in  an  acid 
coagulation  of  the  casein.  The  degree  of  acidity  is  usually  sufficient 
to  inhibit  the  development  of  proteolytic  bacteria  and  also  a  majority 
of  pathogenic  bacteria  as  well.  Occasionally  other  types  of  fecal 
bacteria  may  be  isolated  from  milk.  Of  these  Bacillus  coli  has 
received  much  attention,  chiefly  through  its  constant  association  with 
human  as  well  as  with  bovine  excrement.  Papasotirin  and  Prescott2 
have  isolated  bacteria  indistinguishable  from  Bacillus  coli  by  cultural 
methods  from  hay  and  dried  grains  and  the  organism  is  very  frequently 
present  in  flour,  consequently  the  identification  of  it  in  milk  does  not 
furnish  conclusive  evidence  of  contamination  either  from  human  or 
bovine  sources.  Bacillus  coli  does  not  produce  more  than  minimal 
amounts  of  gas  in  milk,  although  its  aerogenic  activity  in  dextrose  and 
lactose  broth  is  one  of  its  noteworthy  cultural  cMaracters.  It  does, 
however,  form  sufficient  acid  from  lactose  to  cause  an  acid  coagulation 
of  the  casein.  In  this  respect  it  does  not  differ  markedly  from  other 
lactic  acid  bacteria.  Occasionally,  in  association  with  a  strongly 
proteolytic  bacterium,  as  certain  strains  of  Bacillus  mesentericus,  a 
deep-seated  change  is  brought  about  in  milk  by  the  combined  action 
of  the  two  organisms.  Bacillus  mesentericus  acting  alone  liquefies 

1  Kruse,  Centralbl.  f.  Bakt.,  1903,  I.  Abt.,  xxxiv,  737;    Heinemann,  Jour.  Inf.  Dis., 
1906,  iii,  173. 

2  Centralbl.  f.  Bakt.,  Ref.,  1903,  xxxiii,  279. 


608  BACTERIOLOGY  OF  MILK 

the  casein;  in  symbiosis  with  Bacillus  coli  not  only  are  the  protein 
constituents  of  the  milk  thoroughly  decomposed — a  large  volume  of 
gas  is  formed  as  well  and  the  milk-sugar  is  converted  into  carbon 
dioxide,  hydrogen  and  lactic  acid.1  The  alkaline  products  of  putre- 
faction formed  by  Bacillus  mesentericus  neutralize,  to  a  large  degree, 
the  acid  products  formed  by  Bacillus  coli  and  the  net  change  in  the 
chemical  composition  of  the  milk  is  much  greater  than  the  sum  of 
their  separate  activities. 

Abnormal  bacterial  fermentations  of  milk  are  occasionally  sources 
of  great  trouble  to  dairymen.  One  of  the  more  common  of  these  is 
known  as  ropy  or  shiny  milk,  in  consequence  of  the  viscidity  which 
develops.  Several  kinds  of  bacteria  cause  ropiness,  but  of  these 
Bacillus  lactis  viscosus  appears  to  be  more  frequently  concerned.  A 
bitter  flavor  may  be  imparted  to  milk  either  from  the  feed  of  the  cow 
or  by  the  growth  of  bacteria.  The  latter  is  usually  due  to  the  partial 
digestion  of  the  milk  proteins  resulting  in  an  accumulation  of  pep- 
tones. The  gas  bacillus — Bacillus  aerogenes  capsulatus — produces 
an  energetic  fermentation  of  milk-sugar  and  eventually  a  rather  deep- 
seated  digestion  of  the  casein  if  its  activity  is  not  restricted.  The  spores 
of  the  organism  are  very  resistant  to  physical  agents  and  are  often 
found  in  commercial  grades  of  lactose,  which  is  prepared  from  milk. 
There  is  evidence  that  this  organism,  transmitted  through  milk,  may 
incite  mild  or  severe  diarrhea  in  children,  less  frequently  in  adults. 
Pasteurized  milk,  particularly  that  originating  in  unclean  dairies, 
occasionally  contains  considerable  numbers  of  gas  bacilli  and  the 
absence  of  lactic-acid-forming  bacteria  in  such  milk  (which  normally 
restrain  their  activity)  may  be  a  factor  in  its  ability  to  develop  rapidly. 

Proteolytic  bacteria,  particularly  spore-forming  varieties  of  the 
Subtilis-Mesentericus  Group,  decompose  milk  proteins  with  the  forma- 
tion of  casein  peptones  or  even  polypeptids.  They  occasionally  mul- 
tiply rapidly  in  pasteurized  milk,  when  the  degree  of  heat  applied  has 
been  sufficient  to  kill  the  lactic-acid-producing  bacteria;  ordinarily 
lactic  acid  restrains  the  growth  of  proteolytic  bacteria. 

Pathogenic  bacteria,  as  a  rule,  produce  very  little  change  in  the 
appearance  of  milk  and  the  chemical  composition  also  is  not  greatly 
altered  during  their  development.2  Ordinarily  it  is  impracticable  to 
search  for  pathogenic  bacteria  in  this  medium,  for  the  chances  of 
success  are  minimal. 

.  1  Kendall,  Boston  Med.  and  Surg.  Jour.,  1910,  clxiii,  322. 
2  Kendall,  Day,  and  Walker,  Jour.  Am.  Chem.  Soc.,  1914,  xxxvi,  1937-1966. 


MILK  AND  ITS  RELATION   TO   THE  PUBLIC  HEALTH        609 

Milk  and  Its  Relation  to  the  Public  Health. — The  importance  of 
milk  as  a  medium  for  the  transmission  of  pathogenic  bacteria  is  shown 
in  the  following  list  transcribed  from  the  compilation  of  Trask.1 
Statistics  of  317  epidemics  of  typhoid  fever,  125  epidemics  of  scarlet 
fever,  51  epidemics  of  diphtheria  and  7  of  septic  sore  throat  are  set 
forth  therein.  This  list  is  by  no  means  regarded  as  complete;  it 
includes  only  those  epidemics  of  recent  years  in  which  satisfactory 
evidence  of  the  origin  and  spread  of  disease  is  available. 

Milk  that  is  free  from  frankly  pathogenic  microorganisms  is  not 
necessarily  a  suitable  food  for  man;  it  may  be  deadly  for  young 
children  and  infants.  In  the  past  little  was  definitely  known  of  the 
relation  of  market  milk  to  the  high  death  rate  among  children,  although 
a  very  direct  connection  was  suspected.  Park  and  Holt,  however, 
made  an  extensive  study  of  this  very  important  question  and  their 
results  are  illuminating.  Their  plan  was  to  feed  ten  groups  of  children 
with  milk  of  known  origin;  this  milk  was  mixed  to  secure  uniformity 
and  divided  into  ten  portions.  One-half,  containing  about  1,200,000 
bacteria  per  cubic  centimeter  at  the  time  of  feeding,  was  distributed 
to  one  group;  the  other  half  was  pasteurized  before  delivery.  It 
contained,  on  the  average,  about  50,000  viable  bacteria  per  cubic 
centimeter.  The  observations  were  carried  on  during  the  three 
warmest  months  of  the  year.  Within  a  week  nearly  two-thirds  of  the 
infants  fed  with  raw  milk  developed  mild  or  severe  diarrhea;  about 
25  per  cent,  remained  well.  Of  those  receiving  pasteurized  milk  about 
25  per  cent,  developed  diarrhea  and  75  per  cent,  remained  well.  A 
similar  experiment  was  made  the  following  summer.  Their  conclu- 
sions were:2 

"1.  During  cool  weather,  neither  the  mortality  nor  the  health  of 
the  infants  observed  in  the  investigation  was  appreciably  affected  by 
the  quality  of  the  market  milk  or  by  the  number  of  bacteria  which 
it  contained.  The  different  grades  of  milk  varied  much  less  in  the 
amount  of  bacterial  contamination  in  winter  than  in  summer,  the 
store  milk  averaging  only  about  750,000  bacteria  per  cubic  centimeter. 

"2.  During  hot  weather,  when  the  resistance  of  the  children  was 
lowered,  the  kind  of  milk  taken  influenced  both  the  amount  of  illness 
and  the  mortality;  those  who  took  condensed  milk  and  cheap  store 
milk  did  the  worst  and  those  who  received  breast  milk,  pure  bottled 
milk  and  modified  milk  did  the  best.  The  effect  of  bacterial  contam- 

1  Bulletin  41  of  the  Hygienic  Laboratory,  Washington,  D.  C.,  January,  1908. 

2  Park  and  Holt,  Arch.  Pediat.,  December,  1903,  881. 

39 


610  BACTERIOLOGY  OF  MILK 

ination  was  very  marked  when  the  milk  was  taken  without  previous 
heating;  but  unless  the  contamination  was  very  excessive,  only  slight 
when  heating  was  employed  shortly  before  feeding. 

"3.  The  number  of  bacteria  which  may  accumulate  before  milk 
becomes  noticeably  harmful  to  the  average  infant  in  summer  differs 
with  the  nature  of  the  bacteria  present,  the  age  of  the  milk  and  the 
temperature  at  which  it  has  been  kept.  When  the  milk  is  taken  raw, 
the  fewer  the  bacteria  present  the  better  are  the  results.  Of  the 
usual  varieties,  over  1,000,000  bacteria  per  cubic  centimeter  are  cer- 
tainly deleterious  to  the  average  infant.  However,  many  infants 
take  such  milk  without  apparently  harmful  results.  Heat  above  170° 
F.  (77°  C.)  not  only  destroys  most  of  the  bacteria  present,  but,  appa- 
rently, some  of  their  poisonous  products.  No  harm  from  the  bacteria 
previously  existing  in  recently  heated  milk  was  noticed  in  these 
observations  unless  they  had  amounted  to  many  millions,  but  in  such 
numbers  they  were  decidedly  deleterious. 

"4.  When  milk  of  average  quality  was  fed,  sterilized  and  raw,  those 
infants  who  received  milk  previously  heated  did,  on  the  average, 
much  better  in  warm  weather  than  those  who  received  it  raw.  The 
difference  was  so  quickly  manifest  and  so  marked  that  there  could  be 
no  mistaking  the  meaning  of  the  results. 

U5.  No  special  varieties  of  bacteria  were  found  in  unheated  milk, 
which  seemed  to  have  any  special  importance  in  relation  to  the  summer 
diarrhea  of  children.  A  few  cases  of  acute  indigestion  were  seen  imme- 
diately following  the  use  of  pasteurized  milk  more  than  thirty-six 
hours  old.  Samples  of  such  milk  were  found  to  contain  more  than 
100,000,000  bacteria  per  cubic  centimeter,  mostly  spore-bearing 
varieties.  The  deleterious  effects,  though  striking,  were  neither  serious 
nor  lasting. 

"6.  After  the  first  twelve  months  of  life,  infants  are  less  and  less 
affected  by  the  bacteria  in  milk  derived  from  healthy  cattle.  Accord- 
ing to  these  observations,  when  the  milk  had  been  kept  cool,  the 
bacteria  did  not  appear  to  injure  the  children  over  three  years  of  age 
at  any  season  of  the  year,  unless  in  very  great  excess. 

"7.  Since  a  large  part  of  the  tenement  population  must  purchase 
its  milk  from  small  dealers,  at  a  low  price,  everything  possible  should 
be  done  by  health  boards  to  improve  the  character  of  the  general  milk 
supply  of  cities  by  enforcing  proper  legal  restrictions  regarding  its 
transportation,  delivery  and  sale.  Sufficient  improvements  in  this 
respect  are  entirely  feasible  in  every  large  city,  to  secure  to  all  a  milk 


MILK  AND  ITS  RELATION  TO   THE  PUBLIC  HEALTH        611 

.which  will  be  wholesome  after  heating.  The  general  practice  of 
heating  milk,  which  has  now  become  a  custom  among  the  tenement 
population  of  New  York,  is  undoubtedly  a  large  factor  in  the  lessened 
infant  mortality  during  the  hot  months. 

"8.  Of  the  methods  of  feeding  now  in  vogue,  that  by  milk  from 
central  distributing  stations  unquestionably  possesses  the  most 
advantages,  in  that  it  secures  some  constant  oversight  of  the  child 
and,  since  it  furnishes  the  milk  in  such  a  form  that  it  leaves  the 
mother  least  to  do,  it  gives  her  the  smallest  opportunity  of  going 
wrong.  This  method  of  feeding  is  one  which  deserves  to  be  much 
more  extensively  employed  and  might,  in  the  absence  of  private  philan- 
thropy, wisely  be  undertaken  by  municipalities  and  continued  for 
the  four  months  from  May  15  to  September  15. 

"9.  The  use,  for  infants,  of  milk  delivered  in  sealed  bottles,  should 
be  encouraged  whenever  this  is  possible,  and  its  advantage  duly 
explained.  Only  the  purest  milk  should  be  taken  raw,  especially  in 
summer. 

"10.  Since  what  is  needed  most  is  intelligent  care,  all  possible  means 
should  be  employed  to  educate  mothers  and  those  caring  for  infants, 
in  proper  methods.  This,  it  is  believed,  can  most  effectively  be  done 
by  the  visits  of  properly  qualified  trained  nurses  or  women  physicians 
to  the  homes,  supplemented  by  the  use  of  printed  directions. 

'11.  Bad  surroundings,  though  contributing  to  bad  results  in  feeding, 
are  not  the  chief  factors.  It  is  not,  therefore,  merely  by  better  housing 
of  the  poor  in  large  cities  that  we  will  see  a  great  reduction  in  infant 
mortality. 

"12.  While  it  is  true  that  even  in  tenements  the  results  with  the  best 
bottle  feeding  are  nearly  as  good  as  average  breast  feeding,  it  is  also 
true  that  most  of  the  bottle  feeding  is  at  present  very  badly  done; 
so  that,  as  a  rule,  the  immense  superiority  of  breast  feeding  obtains. 
This  should,  therefore,  be  encouraged  by  every  means  and  not  dis- 
continued without  good  and  sufficient  reasons.  The  time  and  money 
required  for  artificial  feeding,  if  expended  by  the  tenement  mother  to 
secure  better  food  and  more  rest  for  herself,  would  often  enable  her 
to  continue  nursing  with  advantage  to  her  child. 

"13.  The  injurious  effects  of  table  food  to  infants  under  a  year  old, 
and  of  fruits  to  all  infants  and  young  children  in  cities,  in  hot 
weather,  should  be  much  more  generally  appreciated." 

These  observations  do  not  correlate  the  incidence  of  diarrhea  with 
specific  microorganisms,  but  they  do  furnish  strong  presumptive 


612  BACTERIOLOGY  OF  MILK 

evidence  of  the  relative  salubriety  of  milk  containing  small  numbers 
of  bacteria.  The  importance  of  a  consistently  low  bacterial  content 
in  milk  designed  for  human  consumption  has  been  generally  recog- 
nized by  city,  state  and  national  health  bureaus,  and  the  grading  and 
control  of  public  milk  supplies  has  been  one  of  the  great  hygienic 
questions  of  the  last  decade.  The  older  conception  of  a  chemical 
standard  to  safeguard  the  financial  interest  of  the  consumer  has  been 
broadened  to  include  a  bacteriological  standard  which  aims  to  exclude 
milk  containing  an  excessive  number  of  bacteria  from  the  public 
market.  The  bacterial  standard  adopted  varies  somewhat  in  different 
cities,  but  in  general  it  is  so  defined  that  all  milk  which  meets  its 
requirements  must  of  necessity  be  produced  in  clean  dairies,  handled 
carefully  and  consistently  maintained  at  a  low  temperature.  The 
bacterial  standard  is  based  upon  the  number  of  bacteria  per  cubic 
centimeter  of  milk  and  it  is  rapidly  becoming  a  custom  to  recognize 
grades  of  milk,  each  of  which  must  conform  to  certain  regulations 
regarding  production,  handling  and  bacterial  count. 

Certified  milk  is  the  hygienic  grade  milk.  It  is  usually  the  product 
of  a  single  dairy;  the  cows  must  be  free  from  tuberculosis  or  other 
disease  and  stringent  regulations  for  the  condition  of  the  entire  plant 
are  laid  down.  The  milk  as  delivered  must  contain  less  than  the 
maximum  number  of  bacteria  per  cubic  centimeter,  as  set  forth  in 
the  standard.  Usually  the  standard  specifies  10,000  to  30,000  bacteria 
per  cubic  centimeter.  Certified  milk  is  usually  safe  milk,  but  con- 
tamination of  it  with  human  pathogenic  organisms  is  not  at  all 
impossible.  Ordinary  market  milk  is  produced  under  less  rigorous 
conditions  and  the  bacterial  content  is  usually  much  greater;  from 
100,000  to  500,000  bacteria  per  cubic  centimeter,  or  even  1,000,000 
bacteria  represent  the  usual  standards  enforced. 

Pasteurization  of  milk  is  rapidly  becoming  obligatory  in  many 
cities,  particularly  for  the  ordinary  grades  of  milk.  Pasteurization  is 
carried  out  by  heating  milk  to  about  145°  F.  (the  degree  of  heat 
varies  in  different  places),  and  maintaining  it  at  that  temperature  for 
thirty  minutes.  This  degree  and  duration  of  heat  is  deemed  sufficient 
to  weaken  or  destroy  pathogenic  organisms  without  altering  the 
nutritive  value.  The  ideal  method  of  pasteurization  is  to  heat  the 
milk  to  the  required  temperature  for  the  required  time  in  the  bottle 
which  goes  to  the  consumer,  thus  entirely  eliminating  the  danger  of 
human  contamination  subsequent  to  the  process. 

The  pasteurizing  process  does  not  kill  many  of  the  milk  bacteria; 


CELLULAR   ELEMENTS  OF  MILK  613 

thus  Ayers  has  shown  that  an  exposure  of  thirty  minutes  at  a  tem- 
perature of  145°  C.  fails  to  kill  all  colon  bacilli.1  The  bacteria  which 
survive  pasteurization  at  this  temperature  are  chiefly  acid  formers.2 

Cellular  Elements  of  Milk. — It  has  long  been  known  that  milk 
drawn  from  healthy  cows  contains  variable  numbers  of  cellular  ele- 
ments; these  elements  have  been  variously  referred  to  as  leukocytes, 
milk  leukocytes,  pus  cells  or  gland  cells.  They  may  be  either  mono- 
nuclear  or  polymorphonuclear,  and  there  is  little  unanimity  in  inter- 
preting their  significance.  Harris3  believes  they  have  little  sanitary 
significance  as  a  general  rule.  Attempts  have  been  made  to  correlate 
the  numbers  of  cellular  elements  in  milk  with  the  leukocyte  and  eryth- 
rocyte  count  of  the  blood  of  the  homologous  animal,  but  without 
avail.4 

It  is  a  fact,  however,  that  an  inflammation  of  the  udder  of  the  cow 
is  frequently  associated  with  an  unusually  large  number  of  cells  in 
the  milk,  indistinguishable  from  polymorphonuclear  leukocytes,  and 
at  times  these  cells  are  phagocytic.  The  increase  in  cellular  content 
may  be  definitely  restricted  to  one  quarter  of  the  udder. 

An  examination  of  the  milk  freshly  drawn  from  168  normal  cows 
was  made  quantitatively  for  cellular  elements  and  over  80  per  cent, 
of  the  animals  (composite  sample  from  four  quarters  of  the  udder) 
showed  less  than  400,000  cells  per  cubic  centimeter  of  milk.  The 
period  of  lactation  appeared  to  exercise  little  influence  upon  the 
cellular  content,  provided  the  samples  were  collected  at  least  two 
weeks  after  parturition.5 

1  Jour.  Agr.  Res.,  1915,  iii,  No.  5. 

2  Ayers  and  Johnson,  Bull.  126,  Bureau  Animal  Industry,  1910;  ibid,  Bull.  161,  1913. 

3  Jour.  Inf.  Dis.,  1907,  Supp.  Ill,  p.  50. 

4  At  present  comparatively  little  attention  is  directed  to  the  cellular  content  of  milk, 
and  inasmuch  as  it  is  usually  impossible  to  trace  the  milk  to  its  source  after  it  is  bottled 
in  the  city,  the  method  is  not  of  much  practical  importance.     A  careful  histological 
study  of  the  cellular  elements  of  milk  by  a  competent  cytologist  might  reasonably  be 
expected  to  throw  at  least  some  light  upon  the  origin  and  significance  of  milk  leukocytes. 

5  Kendall,  Collected  Studies  from  the  Research  Laboratory,  New  York  City,  iii,  169. 


CHAPTER  XXXII. 
BACTERIOLOGY  OF  THE  SOIL,  WATER,  AND  AIR. 

SOIL. 

THE  upper  layers  of  the  soil  in  arable  regions  of  the  Torrid  and 
Temperate  Zones  are  densely  populated  with  bacteria,  many  of  which 
occur  with  such  regularity  that  they  are  properly  regarded  as  the 
normal  bacterial  flora  of  the  soil.  Others  are  of  transitory  or  accidental 
occurrence,  reaching  the  soil  from  the  air,  from  water,  from  excrement 
and  other  waste  products  of  man  and  animals,  and  from  the  dead 
bodies  of  man,  animals,  and  plants. 

The  very  uppermost  layer  of  the  soil,  the  first  two  or  three  centi- 
meters, which  is  exposed  to  sunlight  and  frequent  desiccation,  usually 
contains  fewer  bacteria  than  the  next  layer,  from  15  to  20  cm.  in  depth. 
Here  the  bacterial  population  is  enormous,  frequently  reaching  several 
millions  of  organisms  per  gram  earth.  Below  this  level  the  number 
of  microorganisms  diminishes  rapidly,  as  Fraenkel1  showed  many 
years  ago.  At  a  depth  of  from  one  to  two  inches  in  undisturbed  soil 
the  bacterial  flora  is  relatively  insignificant  in  numbers  and  frequently 
no  microorganisms  are  found. 

The  character  of  the  soil  and  its  state  of  cultivation  are  reflected  in 
the  bacterial  population  which  will  develop  upon  ordinary  media. 
Thus  sandy  soil  may  contain  but  a  few  hundred  thousand  organisms.2 
Actively  cultivated  soils  frequently  contain  one  to  several  millions  of 
bacteria.3  Soil  permanently  covered  with  grass  is  usually  relatively 
poor  in  bacteria.4  The  dust  of  streets  may  contain  from  one  to  ten 
million  bacteria  per  gram,5  and  soil  intimately  contaminated  with 
manure  may  exhibit  as  many  as  78,000,000  bacteria  per  gram.6  It 
is  not  surprising,  from  these  figures,  to  find  that  the  fertility  of  the 
soil  is  closely  related  to  its  bacterial  population.  Normal  fertile  soils 

*  Ztschr.  f.  Hyg.,  1887,  ii,  521. 

2  Adametz,  Untersuch.  ii.  niederen  Pilze  der  Akerkrume,  1886. 

3  Chester,  Delaware  Agr.  College  Expt.  Station  Report,  1900-1901. 

4  Chester,  Bacteria  of  the  Soil,  etc.,  Bull.  No.  98,  U.  §.  Dept.  of  Agriculture. 

5  Manfredi,  Atti  della  R.  Acad.  della  Science  di  Napoli,  1891,  ii. 

6  Maggiora,  Roy.  Accad.  di  Medicina,  1897,  No.  3. 


SOIL  615 

contain  large  numbers  of  microorganisms,  and  sand,  which  is  notor- 
iously infertile,  contains  relatively  few. 

The  normal  bacterial  flora  of  fertile  soil  consists  essentially  of  at 
least  two  distinct  types  of  organisms;  they  may  be  classified  accord- 
ing to  their  chemical  activity  into  those  which  effect  a  rapid  deep- 
seated  decomposition  of  dead  organic  matter  into  simple  combinations 
of  the  elements  which  enter  into  its  composition — ammonia,  carbon 
dioxide,  hydrogen  sulphide,  and  so  on — and  those  which  transform 
these  simple  compounds,  especially  ammonium  salts,  into  nitrites  and 
eventually  into  fully-oxidized  (mineralized)  nitrates.  In  the  latter 


FIG.  98. — Bacillus  subtilis  showing  spores.    X  1000. 

form  the  nitrogen  originally  present  in  organic  matter  is  available  for 
plant  synthesis  into  protein  through  the  action  of  sunlight  upon  the 
chlorophyll  of  the  vegetable  kingdom,  thus  completing  the  cycle. 

The  initial  phase  in  the  degradation  of  dead  organic  matter  to 
ammonium  salts  and  simple  compounds  of  the  other  elements  which 
comprise  the  protein  molecule  appears  to  be  accomplished  largely 
through  the  activity  of  bacteria  of  the  Subtilis-Mesentericus  and  Proteus 
Groups.  These  organisms  elaborate  powerful  active  soluble  proteo- 
lytic  enzymes  which  liquefy  protein,  and  eventually  the  intracellular 
digestion  of  the  hydrolytic  cleavage  products  of  protein  by  these 
microorganisms  results  in  ammonia  formation.1 

The  Proteus  Group  has  been  discussed  elsewhere.2  The  cultural 
characters  of  the  Subtilis-Mesentericus  Group  are  as  follows: 

1  Kendall,  Day  and   Walker,  Jour.  Am.  Chem.  Soc.,  1913,  xxxv,  1243;   ibid.,   1914, 
xxxvi,  1966;  Jour.  Inf.  Dis.,  Npvember,  1915. 

2  Page  359.  - 


616          BACTERIOLOGY  OF  THE  SOIL,   WATER,  AND  AIR 

Morphology. — Rod-shaped  organisms  with  rounded  ends,  occurring 
usually  in  chains  of  greater  or  lesser  length.  The  individual  cells 
measure  from  0.7  to  1.2  microns  in  diameter,  and  vary  in  length  from 
2.5  to  9  microns.  The  members  of  the  group  are  actively  motile  prior 
to  sporulation  and  possess  numerous  peritrichic  flagella.  No  capsules 
are  formed,  but  spore  formation  is  a  characteristic  feature  of  the  group. 
The  morphological  details  of  spore  formation  and  spore  germination 
are  relied  upon  largely  to  distinguish  the  various  members  of  the 
group,  but  these  details  are  of  no  practical  significance  in  this  dis- 


cussion. 


Isolation  and  Culture. — The  organisms  of  the  Subtilis-Mesentericus 
Group  grow  with  great  luxuriance  upon  ordinary  cultural  media.  The 
colonies  on  agar  are  irregular  in  shape,  opaque,  and  spread  rapidly. 
Gelatin  colonies  are  similar  in  appearance  and  the  medium  is  rapidly 
liquefied.  Blood  serum  and  casein  are  also  liquefied.  Milk  is  coagu- 
lated and  the  coagulum  dissolves;  the  reaction,  at  first  slightly  acid, 
soon  becomes  alkaline  as  a  rule.  Indol,  ammonia  in  considerable 
amounts,2  hydrogen  sulphide  and  other  products  of  protein  decom- 
position are  formed  in  dextrose-free  media  and  cultures  of  the  organisms 
contain  very  powerful  soluble  proteases.  The  addition  of  dextrose 
to  such  media  definitely  prevents  the  formation  of  such  proteases, 
however.3 

As  a  rule  the  Subtilis-Mesentericus  bacilli  are  non-pathogenic,  but 
Silberschmidt4  and  others  have  described  a  type  of  ophthalmia  in 
Switzerland,  apparently  incited  by  Bacillus  subtilis,  and  Spiegelberg,5 
Fliigge,6  Ardoin7  and  more  recently  Vincent8  have  presented  evidence 
in  favor  of  the  view  that  the  organisms  may  become  temporarily 
localized  in  the  intestinal  tract  and  incite  severe  gastro-intestinal 
disturbances. 

It  is  stated  that  Bacillus  subtilis  differs  from  Bacillus  mesentericus 
and  other  members  of  the  group  in  its  inability  to  ferment  dextrose. 
The  other  varieties  form  acid  but  no  gas  from  this  sugar. 

The  foregoing  observations  have  shown  that  the  normal  bacterial 
flora  of  the  soil  plays. a  prominent  part  in  agriculture;  it  transforms 
dead  unavailable  organic  matter  and  certain  minerals  as  well  into 

1  Gottheil,  Centralbl.  f.  Bakt.,  1901,  vii,  II  Abt.     Arthur  Meyer,  Practicum  d.  botan- 
ischen   Bakterienkunde,   Jena,    1903.     Chester,    Delaware   College   Agricultural   Expt. 
Station,  Ann.  Kept.,  1902-1903. 

2  Kendall,  Day  and  Walker,  loc.  cit.  3  Kendall  and  Walker,  loc.  cit. 

4  Ann.  Inst.  Past.,  1903,  xvii,  268.  5  Jahrb.  f.  Kinderheilk.,  1899,  xlix,  194. 

6  Ztschr.  f.  Hyg.,  1894,  xvii,  272.  "  These  de  Paris,  1898,  p.  78. 

8  Intestinal  Toxemia  in  Infants,  1911. 


SOIL  617 

compounds  suitable  for  plant  food.  It  is  essential  to  relate  in  some 
detail  the  manner  in  which  these  transformations  are  accomplished. 

The  amount  of  nitrogen  available  at  the  present  time  for  synthesis 
by  plants  exists  chiefly  in  an  organized  state,  and  as  nitrates  in  the 
soil.  Nitrates  are  very  soluble  and  it  is  obvious  that  large  amounts 
of  available  nitrogen  are  yearly  carried  in  solution  to  the  ocean  where 
they  are  practically  lost.  Brandt1  estimates  this  loss  to  be  about 
40,000,000  kilograms  annually.  It  is  obvious  that  this  loss  must  be 
compensated  for. 

It  is  a  matter  of  common  observation  that  soil  left  uncultivated 
gains  in  fertility  from  year  to  year  and  in  1875  Barthelot,  and  Nobbe 
and  Hiltner2  made  the  important  discovery  that  nitrogen  from  the 
air  is  fixed  in  the  soil.  It  was  found  that  soil  heated  to  100°  C.  lost 
its  power  of  fixation  of  nitrogen,  suggesting  that  microorganisms 
played  a  part  in  the  process.  In  1888  Beijerinck3  made  the  very 
important  discovery  that  nodules4  upon  the  roots  of  leguminous 
plants  contain  pleimorphic  organisms,  Bacillus  radicicola,  which 
were  able  to  fix  atmospheric  nitrogen.  Maze5  and  others  have  con- 
firmed this  observation!  Somewhat  later  Winogradsky6  isolated  an 
anaerobic  spore-forming  bacillus,  Clostridium  pasteurianum,  not 
depending  upon  plants  for  its  sustenance,  but  free  living,  which 
accomplished  the  same  transformation,  and  in  1901  Beijerinck7  iso- 
lated and  described  the  very  important  group  of  Azobacteria,  which 
are  widely  distributed  in  the  soil  and  are  able  to  fix  atmospheric 
nitrogen.  These  organisms  are  most  active  when  associated  with 
other  soil  bacteria,  but  are  fully  able  to  fix  nitrogen  when  grown  in 
pure  culture  in  artificial  media  free  from  nitrogenous  compounds. 

The  oxidation  of  ammonia  salts  to  nitrites  and  then  to  nitrates  is 
effected  through  the  activities  of  nitrifying  bacteria,  first  isolated  and 
described  by  Warrington  and  Winogradsky.  Two  organisms  are 
concerned,  a  coccus,  Nitrosococcus,  which  transforms  ammonium 
salts  to  nitrites,  and  a  small  bacillus,  Nitrobacter,  which  oxidizes 
nitrites  to  nitrates.  These  organisms  do  not  thrive  in  the  presence 
of  complex  organic  matter  and  appear  to  derive  their  nutritive  require- 

1  Report  Kommission  zur  Untersuch.  d.  deutsche  Meere,  1899-1901. 

2  Landwirthsch.  Versuchsstat,  xlv. 

3  Bot.  Zeitung,  1888,  725. 

4  These  nodules  were  first  described  by  Hellriegel   (Tageblatt  Naturforsch.   Vers., 
Berl.,  1886,  290)  and  Willforth  (ibid.,  1887,  362). 

6  Ann.  Inst.  Past.,  1897,  xi;    1898,  xii. 

6Compt.  rend.  Soc.  biol.,  1893,  cxvi,  1385;    1894,  cxviii,  353. 

7  Centralbl.  f.  Bakt.,  1901,  vii,  562,  II  Abt. 


618          BACTERIOLOGY  OF  THE  SOIL,   WATER,  AND  AIR 

ments  chiefly  from  inorganic  salts.  The  nitrates  are  taken  up  by 
chlorophyll-bearing  plants  and,  with  the  energy  of  sunlight  transform 
them,  together  with  carbon  dioxide,  water,  phosphates  and  various 
salts,  into  the  complex  vegetable  proteins  upon  which  the  animal 
kingdom  primarily  subsists. 

It  is  obvious,  therefore,  that  there  is  a  well-defined  nitrogen  cycle — 
an  intricate  series  of  changes  which  proteins  and  their  derivatives 
undergo,  through  which  complex,  lifeless  nitrogenous  compounds 
are  reduced  through  bacterial  activity  to  simple,  stable  mineralized 
inorganic  combinations  of  their  elements.  These  elements  are 
restored,  chiefly  through  the  synthetic  activity  of  plant  life,  to  the 
animal  kingdom.  The  nitrogen  cycle  is,  in  a  sense,  a  measure  of  the 
metabolism  of  the  living  earth,  in  which  the  anabolic  or  synthetic 
processes  occur  in  plants  and  indirectly  in  animals;  the  catabolic  or 
analytic  process  is  brought  about  chiefly  by  bacteria. 

In  addition  to  the  normal  bacterial  flora  of  the  soil  and  adventitious 
saprophytic  organisms,  pathogenic  bacteria  are  occasionally  found; 
Bacillus  typhosus,  dysentery  and  cholera  organisms  and  other  excre- 
mentitious  bacteria  are  occasionally  deposited  on  the  ground  with 
human  excrement.  These  microorganisms  do  not,  as  a  rule,  survive 
prolonged  exposure  to  air,  sunlight  and  other  unfavorable  environ- 
mental vicissitudes,  however.  Certain  spore-forming  bacteria — Bacil- 
lus tetani,  anthrax,  symptomatic  anthrax,  malignant  edema  and 
gas  bacilli  are  very  common  in  certain  places.  These  bacteria,  except 
anthrax,  appear  to  multiply  in  the  intestinal  tracts  of  the  herbivora. 

The  natural  or  biological  degradation  and  mineralization  of  dead 
organic  matter  by  bacterial  activity  in  the  upper  layers  of  the  soil, 
so  essential  to  promote  fertility,  is  of  paramount  importance  in  the 
purification  of  water  and  sewage.  Indeed,  the  essential  features  of 
the  nitrogen  cycle  are  involved  in  both  instances. 

WATER   AND   SEWAGE. 

The  very  general  distribution  of  bacteria  in  the  superficial  layers 
of  the  soil  makes  it  almost  inevitable  that  waters  which  wash  the 
surface  of  the  earth  shall  receive  some  bacteria,  consequently  rivers 
and  smaller  streams,  lakes  and  other  surface  waters  always  contain 
bacteria  and  other  microorganisms.  The  number  of  bacteria  per 
unit  volume,  however,  is  far  less  in  water  than  upon  the  land,  unless 
floods  carry  large  amounts  of  soil  with  adherent  organisms  directly 


WATER  AND  SEWAGE  619 

into  water  courses.  Then  the  bacterial  content  of  the  water  is  greatly 
increased. 

The  bacterial  flora  of  surface  waters  is  normally  considerably 
reduced  by  the  action  of  sunlight — which  is  germicidal  at  a  depth  of 
several  feet  in  quiet,  clear  water — by  dilution,  sedimentation,  oxida- 
tion, and  by  the  activities  of  predatory  aquatic  animals.  The  average 
soil  pollution  of  water  by  surface  contamination  in  sparsely  populated 
drainage  areas  is  not  harmful  to  man,  and  such  waters  would  ordinarily 
be  suitable  for  domestic  use. 

Unfortunately  water  courses  are  convenient  channels  for  the 
removal  of  human  waste,  including  excreta,  and  such  waste  is  poten- 
tially dangerous  because  it  may  contain  pathogenic  bacteria.  Exten- 
sive epidemics  of  water-borne  excrementitious  disease — as  typhoid 
and  cholera — have  focused  attention  upon  the  potential  dangers 
attending  the  use  of  unpurified  surface  water  for  domestic  purposes, 
and  the  statistical  evidence  of  a  reduction  in  the  incidence  of  intes- 
tinal diseases  when  water  supplies  have  been  purified  by  filtration 
or  by  other  methods  is  conclusive  proof  of  the  occasional  transmission 
of  excrementitious  diseases  through  polluted  water. 

Ground  water — from  deep  wells  and  from  springs — is  usually  rela- 
tively free  from  bacteria  unless  surface  pollution  occurs.  The  water 
which  feeds  these  sources  is  filtered  free  from  bacteria  during  its 
passage  through  the  deeper  layers  of  the  soil.  Ground  water  is  not 
extensively  used  for  municipal  supplies  at  the  present  time.  Surface 
waters  furnish  the  principal  available  sources  of  this  commodity  for 
domestic  use,  and  in  thickly  settled  areas  it  has  been  found  necessary 
to  purify  the  water  before  it  is  safe  for  human  consumption. 

The  objects  of  water  purification  are:  To  eliminate  pathogenic 
bacteria,  and  to  reduce  the  dissolved  and  suspended  organic  matter 
to  a  state  of  complete  oxidization  and  mineralization.  It  will  be 
remembered  that  bacteria  of  the  soil  effect  a  mineralization  of  organic 
substances,  and  the  purification  of  water  and  of  sewage,  which  is 
grossly  polluted  water,  is  ordinarily  accomplished  by  a  direct  applica- 
tion of  the  same  natural  process. 

For  convenience  in  operation,  filters  are  constructed  which  are 
essentially  water-tight  basins  (to  prevent  the  entrance  of  extraneous, 
unpurified  water)  containing  underdrains  covered  with  a  layer  of 
sand  of  uniform  size,  from  two  to  four  feet  in  thickness.1  The  under- 

1  The  details  of  structure  and  operation  of  filters  designed  for  the  purification  of 
water  and  sewage  are  beyond  the  scope  of  this  volume. 


620          BACTERIOLOGY  OF  THE  SOIL,   WATER,  AND  AIR 

drains  are  designed  to  remove  the  purified  water  and  they  have  little 
or  nothing  to  do  with  the  actual  process  or  purification.  The  sand 
layer  per  se  has  little  action  in  the  purifying  process;  it  does  not 
strain  out  bacteria,  because  the  spaces  between  the  sand  grains  are 
very  great  compared  with  the  size  of  the  organisms.  The  sand  does 
support  upon  its  upper  surface,  however,  a  thin,  delicate  continuous 
layer  of  microorganisms,  the  Schmutzdecke,  through  which  the 
water  (or  sewage)  passes.  This  layer  is  so  compact  and  so  closely 
matted  together  that  all  suspended  matter  (including  both  pathogenic 
and  non-pathogenic  bacteria)  in  the  supernatant  water  is  strained 
out,  and  the  dissolved  organic  substances  pass  with  the  raw  or  unfil- 
tered  water  through  the  bodies  of  the  microorganisms  which  collec- 
tively comprise  the  Schmutzdecke.  During  this  passage  the  dissolved 
organic  matter  undergoes  the  same  general  degradation  to  nitrates 
and  other  fully-mineralized  products  of  microbic  digestion  that 
organic  substances  in  the  upper  layers  of  the  soil  undergo;  the  puri- 
fication of  water  by  sand  filtration  is,  therefore,  a  catabolic  phase  in 
the  nitrogen  cycle,  brought  about  by  bacterial  activity  precisely  as 
the  mineralization  of  organic  substances  in  the  upper  layers  of  the 
soil  is  a  catabolic  phase  of  the  nitrogen  cycle.  The  final  products  in 
each  case  are  normally  nitrates  and  other  inorganic  salts. 

The  efficiency  of  the  purification  of  water  or  of  sewage  by  the 
method  of  sand  filtration  is  therefore  to  be  measured  chemically  and 
bacteriologically.  Chemically  a  complete  transformation  of  complex 
organic  compounds  (ordinarily  determined  as  albuminoid  and  "free 
ammonia")  to  nitrates  is  an  indication  that  the  digestive  power  of  the 
filter  is  at  par.  Bacteriologically  a  disaupearance  of  all  bacteria 
derived  from  human  or  animal  excrement  and  a  great  reduction  of 
the  total  numbers  of  bacteria  in  the  filtered  water  as  compared  with 
the  unfiltered  water  is  evidence  of  the  bacterial  efficiency  of  the 
filter. 

The  chief  source  of  danger  in  potable  waters  is  bacterial  contamina- 
tion from  human  sources.  A  simple  inspection  of  water  frequently 
fails  to  detect  contamination,  and  even  a  chemical  examination  may 
not  suffice  to  reveal  pollution.  Millions  of  typhoid  bacilli  may  be 
introduced  into  a  liter  of  water  without  inducing  changes  that  could 
be  detected  visually  or  chemically.  The  bacteriological  examination 
of  water,  therefore,  is  from  ten  to  one  hundred  times  more  delicate 
than  the  chemical  examination  as  a  means  of  detecting  contamination 
of  water  with  human  or  animal  waste. 


WATER  AND  SEWAGE  621 

Bacteriological  Examination  of  Water. — A  bacteriological  examina- 
tion of  water  requires  relentless  attention  to  details,  from  the  collec- 
tion of  the  sample  to  its  final  analysis  and  interpretation. 

Collection  of  Sample. — It  must  be  borne  in  mind  that  a  small  volume 
of  water — 100  c.c.  or  less — is  ordinarily  collected  as  a  sample  repre- 
senting thousands  or  millions  of  gallons,  consequently  sampling  is  an 
important  detail  in  the  bacteriological  analysis  of  water.  The  col- 
lecting bottle  must  be  clean  and  sterile,  and  the  site  at  which  the 
sample  is  taken  must  be  representative. 

It  is  customary  to  obtain  a  sample  of  water  from  brooks,  rivers  and 
lakes  at  a  distance  from  the  shore,  and  preferably  samples  from 
different  depths  should  be  taken.  The  bottle  must  be  immersed  below 
the  surface  before  water  is  allowed  to  enter  it,  to  avoid  surface  scums. 
If  water  is  taken  from  faucets  or  pumps  the  sample  should  not  be 
collected  until  a  sufficient  flow  has  been  established  to  make  certain 
that  the  fluid  has  come  directly  from  the  water  mains,  or  from  the 
well  itself. 

As  soon  as  the  sample  has  been  collected  it  should  be  examined; 
frequently  this  is  impracticable,  and  the  bottle  should  be  surrounded 
with  ice  at  once  and  shipped  to  the  laboratory.  Ice  restrains  bacterial 
development  for  some  hours  and  this  maintains  the  sample  at  approxi- 
mately its  original  bacterial  content. 

Bacteriological  Analysis  of  Water. — A  bacteriological  examination 
of  water  ordinarily  includes  a  determination  of  the  numbers  of  bacteria 
wrhich  develop  in  ordinary  nutrient  media  at  20°  C.  and  37°  C.,  a  search 
for  organisms  characteristic  of  the  excrement  of  man  or  animals,  their 
approximate  enumeration,  and  other  tests  which  vary  according  to 
the  source  of  the  sample. 

Counting  Bacteria. — The  counting  of  bacteria  ordinarily  signifies 
the  numbers  of  microorganisms  which  will  grow  on  gelatin  incubated 
at  20°  C.,  and  those  that  develop  on  agar  at  37°  C.  Unpolluted  waters 
usually  contain  relatively  few  bacteria  that  will  grow  at  body  tem- 
perature, consequently  the  gelatin  plate  seeded  with  the  same  volume 
of  water  as  the  agar  plate  will  show  many  more  colonies  than  the 
latter;  polluted  waters  show  a  more  even  distribution  of  types  of 
bacteria  that  grow  respectively  at  20°  C.  and  37°  C. 

The  amount  of  water  to  be  plated  in  gelatin  and  in  agar  depends 
upon  the  source  of  the  sample.  Water  from  deep  wells  and  from 
springs  should  contain  relatively  few  organisms,  and  a  cubic  centi- 
meter of  the  sample  is  usually  "planted."  Surface  waters  almost 


622          BACTERIOLOGY  OF  THE  SOIL,   WATER,  AND  AIR 

invariably  contain  more  bacteria  than  ground  waters;  it  may  be 
necessary  to  dilute  a  cubic  centimeter  of  the  sample  with  99  c.c.  of 
sterile  water  to  obtain  the  requisite  distribution  of  organisms  for  an 
accurate  estimation,  or  even  higher  dilutions  may  be  necessary. 
Grossly  polluted  waters  are  diluted  one  thousand  or  even  ten  thousand 
times  with  sterile  water  before  they  are  plated.  In  any  event,  not 
more  than  200  colonies  or  less  than  50  colonies  should  be  present  in 
the  final  dilution,  for,  experience  has  shown  that  greater  numbers  of 
organisms  materially  restrict  development,  and  fewer  than  fifty 
colonies  upon  a  plate  introduces  an  error  in  dilution. 

Technic  of  Plating. — The  sample  of  water,  diluted  to  the  required 
degree  if  necessary,  is  shaken  vigorously  to  break  up  groups  and 
chains  of  bacteria;  a  cubic  centimeter  of  water  is  then  removed  with 
a  sterile  pipette  into  each  of  two  sterile  Petri  dishes,  being  careful  to 
prevent  contamination. 

A  tube  of  sterile  nutrient  gelatin  (10  c.c.)  previously  melted  and 
cooled  to  42°  C.,  is  then  carefully  poured  over  the  water  in  one  Petri 
dish,  and  melted  nutrient  agar  is  similarly  poured  into  the  other  Petri 
dish.  The  water  and  culture  fluid  are  intimately  mixed  by  carefully 
tilting  the  plates,  and  then  set  aside  to  harden.  The  agar  plate  is 
inverted  after  it  has  hardened  to  prevent  condensation  of  moisture 
upon  the  surface  of  the  medium;  this  procedure  reduces  the  possi- 
bility of  confluence  of  surface  colonies.  The  gelatin  plate  is  not 
inverted. 

Incubation  at  20°  C.  for  the  gelatin  plate  and  37°  C.  for  the  agar 
follows.  The  agar  plate  is  counted  after  forty-eight  hours'  incubation, 
the  gelatin  plate  after  four  days. 

Interpretation  of  Bacterial  Count. — At  best  the  quantitative  estima- 
tion of  bacteria  in  water  and  sewage  is  inexact  and  relative  only. 
The  many  factors  of  error  in  sampling,  lack  of  uniformity  in  media, 
the  difficulties  of  counting  colonies  when  several  hundred  have  grown 
in  one  plate — all  tend  to  reduce  the  accuracy  and  precision  of  the 
method.  Again,  the  normal  difference  in  bacterial  content  between 
ground  waters,  surface  waters  and  polluted  waters  makes  an  inter- 
pretation of  the  bacterial  count  somewhat  difficult.  For  example, 
100  bacteria  per  cubic  centimeter  in  a  deep  well  water  might  have 
greater  sanitary  significance  than  500  bacteria  per  cubic  centimeter 
in  a  surface  water,  where  bacterial  counts  are  almost  invariably  higher. 

Attempts  have  been  made  to  establish  arbitrary  bacterial  standards; 
thus,  waters  containing  less  than  100  bacteria  per  cubic  centimeter 


WATER  AND  SEWAGE  623 

were  formerly  regarded  as  safe  waters;  those  containing  from  100  to 
500  organisms  per  cubic  centimeter  were  regarded  with  suspicion,  and 
those  containing  1000  or  more  organisms  were  pronounced  dangerous 
for  domestic  use.  In  the  abstract  these  standards  are  fictitious;  sur- 
face waters  even  in  uninhabited  districts  may  contain  many  hundreds 
of  bacteria  per  cubic  centimeter  after  rains,  yet  the  bacterial  count 
would  convey  but  little  information  of  the  actual  sanitary  status  of 
the  water.  Successive  bacterial  counts  carried  out  over  long  periods 
of  time,  on  the  other  hand,  are  frequently  of  very  great  value.1 

A  direct  examination  of  water  for  pathogenic  bacteria,  as  the  typhoid 
bacillus,  if  it  were  practicable,  would  be  a  most  satisfactory  method  of 
evaluating  domestic  water  supplies,  for  it  is  the  presence  of  these 
organisms  harmful  to  man  which,  in  the  last  analysis,  makes  water 
containing  them  dangerous  for  human  consumption.  Unfortunately 
it  is  not  practicable,  as  numerous  observers  have  amply  demonstrated, 
to  isolate  pathogenic  organisms  of  this  type  directly  from  water,  and 
there  are  but  few  authentic  records  of  a  successful  cultivation  of  the 
typhoid  bacillus  from  water  supplies  known  to  be  infected,  in  spite 
of  numerous  attempts. 

The  practical  impossibility  of  isolating  pathogenic  bacteria  from 
water  has  led  to  the  development  of  methods  for  the  detection  of  Bacil- 
lus coli  and  organisms  found  practically  constantly  in  human  and 
animal  excrement.  Bacillus  coli  is  somewhat  more  tolerant  of  environ- 
mental conditions  as  they  exist  in  water  than  Bacillus  typhosus,  and 
its  constant  presence  in  fecal  discharges  makes  it  somewhat  more 
effective  as  an  indicator  of  excrementitious  contamination  than  the 
frankly  pathogenic  organisms.  The  simplest  and  in  many  respects 
the  best  method  for  detecting  Bacillus  coli  in  water  is  to  add  graduated 
amounts  of  the  sample  to  be  analyzed — beginning  with  1  c.c.  and 
decreasing  the  amount  one-tenth  in  successive  cultures — to  lactose 
fermentation  tubes.2  A  production  of  gas  within  twenty-four  or 
forty-eight  hours  is  suggestive,  but  not  conclusive  evidence  of  the 
presence  of  the  organism.  If  gas  develops  some  of  the  culture  should 
be  placed  on  Endo  medium,  and  red  colonies  that  develop  are  tested 
for  their  ability  to  produce  acid  and  gas  in  dextrose  and  lactose  media, 
for  indol  production  in  sugar-free  broth,  for  their  action  upon  milk, 

1  For  an  excellent  resume  of  the  subject,  see  the  Bacteriology  of  Surface  Waters  in 
the  Tropics,  Clemesha,  London  and  Calcutta,  1912,  and  Prescott  and  Winslow,  Ele- 
ments of  Water  Bacteriology,  New  York,  1913. 

2  Theobald  Smith,  Notes  on  Bacillus  coli  communis  and  Related  Forms,  Am.  Jour. 
Med.  Sci.,  September,  1895,  283. 


624          BACTERIOLOGY  OF  THE  SOIL,   WATER,  AND  AIR 

and    an   absence   of   liquefaction   in   gelatin.     These   reactions   are 
regarded  as  satisfactory  to  establish  the  identity  of  Bacillus  coli. 

In  some  laboratories  a  direct  plating  of  the  sample  of  water  in  lactose 
litmus  agar  or  upon  Endo  medium  is  practiced,  but  this  procedure  is 
considerably  less  sensitive  than  the  fermentation  enrichment  method 
outlined  above. 

Colon  bacilli  may  occasionally  be  isolated  from  considerable  volumes 
of  water — 10  or  100  c.c. — when  they  cannot  be  detected  with  regularity 
in  1  c.c.  or  less.  Very  little  significance  attaches  to  such  results, 
because  experience  has  shown  that  even  springs  in  uninhabited  regions 
may  occasionally  contain  a  few  colon  bacilli,  derived  probably  from 
chance  contamination  with  the  feces  of  wild  animals.  If,  on  the 
contrary,  colon  bacilli  are  regularly  present  in  a  water  supply  to  such 
an  extent  that  a  cubic  centimeter  of  the  water  gives  a  positive  culture 
in  a  decided  majority  of  attempts,  that  water  is  viewed  with  suspicion. 
If  the  organism  is  regularly  present  in  one-tenth  of  a  cubic  centimeter, 
the  water  is  judged  unfit  or  dangerous  for  human  consumption  until 
it  is  purified. 

Other  organisms  have  from  time  to  time  been  proposed  as  indi- 
cators of  pollution — thus  streptococci  and  gas  bacilli  have  been  studied 
in  this  connection — but  up  to  the  present  time  they  havt 
accepted  as  authoritative  criteria  for  evaluating  the  potability  of 
water  supplies. 

BACTERIA   OF   THE   AIR. 

Bacteria  when  dried  and  attached  to  dust  particles  may  be  wafted 
into  the  air  and  remain  suspended  there  for  considerable  periods  of 
time.  Even  the  gentlest  air  currents  suffice  to  prevent  their  settling 
out.  At  high  altitudes  and  over  large  bodies  of  water  the  bacterial 
population  of  the  ah-  is  very  small  indeed;  over  large  cities  and  cul- 
tivated land  the  number  of  organisms  in  the  air  is  frequent]; 
greater.  Heavy  rains  and  snow  tend  to  remove  bacteria  from  the 
atmosphere,  while  dry  windy  weather  increases  the  aerial  contamina- 
tion. 

Usually  the  more  hardy  organisms  alone  are  found  in  the  air,  but  in 
houses  and  hospitals  pathogenic  bacteria  may  be  detected  occasionally; 
probably  the  extrusion  of  minute  droplets  of  sputum1  containing  these 
organisms  is  a  most  potent  factor  in  air  contamination  by  bacteria. 

1  See  Droplet  Infection,  p.  91. 


BACTERIA   OF  THE  AIR  625 

Several  methods  have  been  proposed  for  the  estimation  of  the  num- 
ber of  bacteria  in  the  air;  that  of  Winslow,1  which  consists  essentially 
in  aspirating  a  definite  volume  of  air  through  two  flasks,  each  of  which 
contains  melted  nutrient  gelatin,  is  the  simplest  and  most  direct. 
Comparatively  little  has  been  accomplished  thus  far  from  a  quantita- 
tive study  of  the  bacterial  population  of  the  air;  it  is  possible  that 
an  attempt  to  isolate  specific  types  of  pathogenic  bacteria  from 
theatres  and  other  places  where  large  numbers  of  people  meet  might 
throw  some  light  upon  certain  features  of  the  air  transmission  of 
bacterial  infections  which  are  not  well  understood  at  the  present  time. 

1  Science,  1908,  xxviii.  28. 


40 


ATJTHOB  INDEX. 


ABBE,  19 

Abderhalden,  137 

Abel,  363,  365 

Achalme,  492 

Achard,  344 

Adametz,  614 

Agramonte,  561 

Albrecht,  409,  412,  416 

Alilaire,  59 

Alvarez,  469 

Amoss,  558,  559 

Anders,  482 

Anderson,   122,  248,  394,  451,  481, 

563,  564 
Andrewes,  272 
Ardoin,  616 
Arloing,  496 
Armand,  258 
Armand-Delille,  436 
Armaud,  380 
Arning,  465 
Arnould,  374 
Aronson,  63,  270 
Arrhenius,  126,  143 
Arthus,  133 
Asakawa,  478 
Ashburn,  576 
Atkinson,  142,  396 
Auclaire,  48 
Auer,  134,  135 
Auerbach,  78,  217 
Avery,  133,  287,  435 
Axerifeld,  424 
Ayers,  613 


B 


BABES,  429 

Bagg,  274,  313,  321,  350,  492,  599 

Bail,  165 

Baillon,  481 

Bainbridge,  347,  349,  350 

Baldwin,  448,  453,  456,  477 

Bandi,  518 

Bang,  382 

Banti,  506 

Banzhaf,  141,  143,  396 

Bar,  362 

Barber,  208 

Barker,  440 


560, 


Bartarelli,  518 

Bartel,  440 

Barthelot,  617 

Bassi,  18 

Bates,  321 

Bauer,  435 

Baumgarten,  437 

Bayon,  464 

Beck,  433,  445 

von  Behring,  20,  139,  388,  398,  437,  503, 

504 

Beijerinck,  617 
Beljaeff,  143 
Bensaude,  344 
Bergell,  440 
Berghaus,  217 
Bernheim,  308,  530 
Berterelli,  570 
Besangon,  425 
Besche,  510 
Besredka,  134,  273,  278,  320,  328,  329, 

333,  507 
Besson,  262 
Bettencourt,  299 
Bezold,  404 
Biedl,  134,  135 
Bienn,  577 
Bienstock,  581 
Billroth,  256 
Biltz,  126 
Blaisot,  570 
Blanchard,  527 
Bloomfield,  406 
Blue,  235 
Blumenthal,  478 
Bockenheimer,  480 
Bolduan,  198,  272,  294 
Bollinger,  536 
Booker,  362 
Bordet,  128,    130,    144,    146,    150,    151, 

153,  154,  273,  420,  422 
Borrel,  480 
Bostrom,  538 
Boulton,  474 
Bowman,  441 
Bradley,  440 
Brandt,  617 
Brault,  528,  530 
Breed,  603 
Breymann,  381 

Brieger,  353,  476,  477,  485,  493,  495,  503 
Brill,  560 
Brinkerhoff,  571,  572 


628 


AUTHOR  INDEX 


Brion,  335,  344 

Briscoe,  440 

Brodmeier,  361 

Brown,  28,  272,  275,  276,  282,  448,  491, 

606 

Bruce,  310 
Bruck,  606 
Brucker,  515,  518 
Bruning,  362 
Brims,  297,  300,  481 
Bruschettini,  419 
Buchner,  130,  150,  153,  476 
Bujwid,  503 
Bumm,  301,  304 
Bundesen,  399 
Bunting,  405,  406 
Burckhardt,  259 
Burri,  519 
Busquet,  577 
Busse,  551 
Butterfield,  284,  285 
Buxton,  258,  335,  339,  350,  355 


CACACE,  258 
Calkins,  571 
Calmette,  480 
Canfora,  482 
Canon,  419 
Cantani,  418 
Carapelle,  63 
Carey,  440 
Carini,  481 
Carle,  472 
Carpano,  325 
Carr6,  261 
Carriere,  435 
Carroll,  561 
Carter,  525,  561 
Casagrandi,  286 
Castellani,  328,  526,  527 
Cathcart,  347 
Catrin,  577 
Cecchetto,  570 
Certes,  47 
Chalmers,  527 
Chamberland,  374,  493,  565 
Chantemesse,  343 
Chapin,  410,  411,  412 
Chapman,  150 
Charrin,  379,  380 
Chauffard,  480 
Cherry,  143 
Chester,  614,  616 
Chowning,  576 
Christensen,  554 
de  Christmas,  303 
Citron,  398 

Clark,  77,  220,  557,  558,  559 
Claudius,  494 

Claypole,  325,  331,  332,  333,  334,   336, 
535 


le  Clef,  130,  166 

Clegg,  464,  533 

Clemesha,  623 

Clerc,  262 

Cohen,  477,  570 

Cohn,  19 

Cole,  87,  101,  274,  281,   284,   285,    287, 

289,  290 

Coleman,  335,  339,  350,  356,  598,  599 
Coley,  277 
Comte,  560 

Conradi,  319,  320,  362,  603 
Conseil,  560 
Conseille,  577 
Corbus,  528,  530 
Cornevin,  496 
Corper,  258,  319,  328,  435 
Councilman,  292,  293,  294,  295,  571 
Courmont,  477 
Craig,  576 
Cramer,  60 
Creite,  482 
Gushing,  350,  587 


DALE,  135 

Darling,  321 

Davaine,  372 

Davis,  426,  554 

Day,  58,  62,  70,  78,  79,  202,  218,  223, 
257,  274,  310,  313,  314,  318,  321,  326, 
328,  338,  344,  350,  354,  355,  356,  358, 
360,  361,  366,  380,  391,  433,  492,  504, 
599,  608,  616 

Dean,  468 

Deelman,  360 

Deist,  447 

Delius,  418 

Deneke,  513 

Denys,  130,  166 

Descos,  438 

Deycke,  464 

Dieudonne,  296,  297,  361,  414 

Distaso,  585 

Dochez,  271,  287,  289 

Doerr,  166,  319,  320,  322 

Dohle,  564 

Dold,  286 

Donitz,  478,  479 

Dopter,  300,  318,  320,  321,  322,  323 

Dorset,  430,  432,  436 

Douglas,  130,  166 

Doyen,  477 

Dreyfuss,  61 

Ducrey,  425 

von  Dungern,  262 

Dunham,  340,  488,  491,  503 

Durham,  143,  339 

von  Dusch,  19 

Dutton,  526 
I  Duyal,  464 
i  Dziergowski,  61 


AUTHOR  INDEX 


629 


E  BERTH,  325 

Edwards,  462 
Ehrenberg,  19 
Ehrlich,  19,  117,  126,  151,  153,  475,  493, 

495 

Eichhorn,  165,  310 
Eichstedt,  546 
von  Eiselsberg,  365 
Eisenberg,  26,  34,  183,  453 
Eisenbrey,  135 
von  Eisler,  478 
Eldridge,  315 
Ellinger,  75 
Elmassian,  400 
Elser,  296,  298,  299 
Emmerich,  353,  381 
Emmering,  257 
Emmerling,  62,  272,  360 
Engel,  261 
von  Ermengem,  484 
Ernst,  430,  440 
Errera,  25 
Escherich,  265,  266,  274,  353,  356,  363, 

386,  581,  582,  583,  584,  585,  589,  590 
Esmeiri,  577 
Ewing,  571 
Eyre,  311 


F 


FABYAN,  382,  383,  384,  460 

Farmer,  79,  218 

Fehleisen,  270 

Feri,  577 

Fermi,  360,  381,  476,  477 

Ferran,  474,  507 

Fiessinger,  440 

Finger,  303,  304 

Finkelstein,  385,  582 

Finkler,  511 

Finlay,  561 

Fisch,  474 

Fischer,  A.,  40,  41,  48,  83 

Fischer,  E.,  70,  218,  220 

Flatau,  477 

Flexner,   296,  297,  298,    300,    315,    322, 

557,  558,  559 
Fliigge,  92,  616 
Fluornoy,  525 
Folin,  218 
Force,  334 
Ford,  313,  587 
Forssman,  486 
Forster,  503 
Foulerton,  533,  536 
Fraenkel,  184,  275,  282,  353,  363,  400, 

419,  422,  431,  488,  614 
Franca,  299 
Francetti,  347 
Francis,  573 
Franzen,  77,  356 
Fraser,  558 
Freymouth,  401 


Fried,  37 

Friedberger,  134,  137 
Friedlander,  363 
von  Frisch,  363,  365,  412 
Fritsche,  412 
Frosch,  563 
Fuhrmann,  51 
Furst,  297 


GABBET,  184,  431 

Gaffky,  264,  325,  326,  414 

Galeotti,  63 

Gamaleia,  450,  513 

Gartner,  344,  436 

Gauss,  28 

Gay,  133,  144,  153,  319,  325,  331,  332, 

333,  334,  336 
Geifel,  436 
Gelasesco,  515,  518 
Gelien,  101,  402 
Gengou,  153,  154,  420,  422 
Gessard,  379 
Gessner,  587 
Ghedini,  419 

Ghon,  303,  304,  308,  409,  412,  416,  417 
Gibsoc,  142,  396 
Giemsa,  185 
Gilbert,  267 
Gilchrist,  551 
Gillespie,  271,  289 
Goadby,  101,  264 
Goldberger,  560,  563,  564 
Goldschmidt,  137 
Goldschneider,  477 
Goodhue,  465 
Gordon,  577 
Gorgas,  562 
Gorini,  504 
Gosio,  504 
Gottheil,  616 
Gottstein,  139 
Gotzl,  435 
Graham,  576 
Gram,  182 
de  Gracdi,  28 
Grandi,  472 
Grassberger,  488,  498 
Grawitz,  546 
Greig,  510 
Griffon,  425 
Grigorjeff,  493 
Grimme,  2S 
Grober,  134 

Gruber,  143,  297,  339,  340 
Gruby,  545 

Grunbaum,  143,  149,  340 
Guarnieri,  571 
Gue*nod,  570 
Guerbet,  183 
Gumprecht,  479 
Giinther,  503,  506 
Guthrie,  101,  402 
Gwyn,  350 


630 


AUTHOR  INDEX 


HAASE,  373 

Haffkine,  414,  507 

Hahn,  130 

Halberstadter,  570 

Hamilton,  246,  249 

Hammerschlag,  61 

Handel,  289,  290,  510 

Hankin,  510 

Hansen,  463 

Harden,  77,  354 

Harris,  528,  613 

Hasterlik,  506 

Hastings,  356 

Hauser,  359 

Hausmann,  217 

Hawthorn,  435 

Heinemann,  391,  607 

Heinze,  62 

Hektoen,  117,   130,   132,   169,   548,   554, 

563 

Hellriegel,  617 
Henderson,  422 
Henri  jean,  475 
Herb,  577 
Herms,  558 

Herter,  585,  590,  596,  597 
Herzog,  570 
Hess,  587 
Hetsch,  335,  348 
Heyse,  482 
Hibler,  585 
Hilgermann,  589 
Hill,  176,  564,  565 
Hiltner,  617 
Himmelberger,  462 
Hirsch,  581 
Hirschfelder,  288 
Hiss,  181,  200,  204,  283,  285 
Hochsinger,  482 
Hodenpyl,  434,  443,  459 
Hoffmann,  481,  514,  518,  521,  528,  529 
Hofmann,  404 
Hohlbeck,  482 
Hohn,  297,  300 
Hollander,  444 
Holmes,  436 
Holt,  609 
Holth,  384 
Hooke,  18 
Horder,  272 
Hornor,  422 
Horvath,  48 
Howard,  271,  492,  558 
Hueppe,  270,  363 
Huntoon,  296,  298,  299 


IRVANOFF,  62 
Isaeff,  286,  506 
Israel,  536 


JACOEITZ,  296 
Jaeger,  293,  299,  362 
Jakowski,  381 
Jenner,  571 
Jobling,  138,  297,  440 
Joest,  441 
Johne,  373 
Johnson,  613 
Jones,  222 
de  Jong,  462 
Jordan,  327,  358 
Joubert,  493 
Jouhaud,  267 
Jundell,  419 
Jungano,  585 


KAMEN,  423 

Kappes,  58 

Karlinski,  470 

Kartulis,  423 

Kayser,  335,  344 

Kedrowski,  464 

Keller,  453 

Kempner,  485,  486,  487 

Kendall,  54,  58,  62,  68,  78,  83,  86,  102, 
103,  110,  182,  202,  210,  217,  218,  222, 
223,  257,  274,  310,  313,  314,  315,  318, 
321,  324,  326,  328,  338,  346,  350,  354, 
355,  357,  358,  360,  361,  362,  364,  366, 
380,  385,  386,  391,  392,  393,  433,  435, 
476,  492,  504,  579,  581,  583,  584,  585, 
586,  590,  595,  597,  599,  600,  608,  613, 
615,  616 

Kersten,  327 

Keysser,  138 

Kirchner,  307 

Kirschbert,  404 

Kitasato,  20,  139,  388,  408,  472,  473,  477 

Kite,  456 

Klebs,  256,  269,  325,  388 

Klein,  344,  429,  432,  488,  492,  553 

Klemperer,  286 

Klimenko,  422 

Klimmer,  385 

Kling,  558 

Klinger,  337 

Knapp,  405,  523,  525 

Knorr,  477,  479 

Kober,  439 

Koch,  19,  269,  275,  280,  372,  423,  429, 
430,  461,  493,  495,  499,  505,  506 

Kolb,  262 

Kolle,  142,  297,  335,  413,  418,  506,  507 

Kolmer,  399 

Kon,  510 

Kopetsky,  295 

Kossel,  409 

Kraus,  134,  135,  149,  319,  320,  322,  502, 
568 

Kresling,  64,  430 


AUTHOR  INDEX 


631 


Krompecher,  28 

Krumwiede,  439,  459,  530 

Kruse,  58,  60,  63,  65,  73,  77,   183,  262, 

267,  288,  289,  315,  323,  581,  607 
Kulescha,  510 
Kupriano,  504,  512 
Kuthy,  446,  448,  452 
Kutscher,  299,  494 


LADENBURG,  75 

Laird,  456 

Lamar,  288 

Lamb,  412 

Landmann,  486 

Landsteiner,  478,  557,  558 

von  Langenbeck,  546 

Lanz,  587 

Larsen,  384 

Latour,  18 

Laveran,  577 

Lazear,  561 

Leach,  63 

Leclainche,  498 

Ledderhose,  380 

von  Leeuwenhoek,  18 

Lehmann,  37,  258,  374 

Leishman,  166 

Lenk,  440 

Lentz,  315,  316,  318 

Lespinasse,  305,  307 

Leuchs,  296,  298,  486 

Leutscher,  232 

Levaditi,  429,  518,  519,  557,  558,  568 

Levene,  62,  430,  477,  478 

Levin,  589 

Levy,  328,  360,  361,  481 

Lewis,  134,  135,  557,  559 

Lewith,  38 

Lewkowicz,  266,  267 

Libman,  581 

Liborius,  494 

Lindenthal,  488 

von  Lingelsheim,  270,  296,  298,  299 

Lippman,  267 

Lipschutz,  439 

Lister,  19 

Livingston,  587 

Loeb,  258,  259 

Loffler,  367,  388,  400,  404,  500,  563 

von  Loghem,  51 

Low,  381 

Lowden,  566,  567 

Lowenstein,  58,  433,  445 

Lubarsch,  436 

Lucas,  557 

Ludke,  319 

Lundstrom,  486 

Lustig,  63 

Lyall,  274 

Lyons,  60 


M 


McCLiNTic,  248 

McCoy,  410,  411,  412,  465,  468 

McDaniel,  389 

McFarland,  481,  573 

McGaffin,  321 

Mclntosh,  336 

McQueen,  336 

MacConkey,  587 

MacFadyen,  286,  328,  384 

Mackie,  525 

Maclsen,  126,  143 

Maggiora,  614 

Magrath,  571 

Mallory,    184,    186,    187,  212,  292,  293, 

294,  295,  329,  422 
Malmsten,  545 
Mandelbaum,  391 
Manfredi,  614 
Manteufel,  525 
Marchand,  166 
Marie,  440,  475,  477,  478 
Marie  esco,  487 
Marmorek,  273,  275,  278,  280 
Martin,  143,  376,  400 
Martini,  315 
Marx,  28 
Massart,  128 
Massea,  359 
Mayer,  183,  297,  348 
Mayerhof,  360 
Maze,  617 
Mecray,  577 
Meier,  156 
Meloy,  435 
Meltzer,  48,  288 
Melvin,  384,  460 
Menzer,  274 
Mereschkowsky,  385 
Metchnikoff,  20,  117,  128,  329,  333,  478, 

504,  506,  512,  517,  589,  596 
Meyer,  27,  29,  62,  274,  479,  616 
Meyerhof,  361 
Mezinescu,  468 
Michaelis,  178,  577 
Mieremet,  405 
Migula,  33 
Miller,  101,  511 
Milne,  526 
M'Leod,  273 
M'Nee,  274 
Moeller,  180,  469,  470 
Mohler,  165,  310,  462 
Moment,  376 
Monvoisin,  433 
Moody,  399 

Moore,  344,  439,  459,  462 
Morax,  400,  424,  475,  477,  478 
Moreschi,  342 
Morgan,  318,  482 
Morgenroth,  151,  153 
Moro,  259,  385,  582,  589 
Moschowitz,  134,  140 
i  Moshage,  399 


632 


AUTHOR  INDEX 


Moss,  101,  402 
Mossu,  433 
Moxter,  151,  153 
Much,  228,  259,  432 
Muhlens,  528 
Miiller,  165,  264,  451 
Musgrave,  320,  533 


N 


NAEGELI,  436 

Nakanishi,  27,  31,  178 

Nashimura,  63 

Nathan,  137 

Neelsen,  184,  431 

Negri,  405,  566 

Neisser,  181,  258,  259,  301,  320,  389,  404, 

463 

Nencki,  58,  74 
Neufeld,  130,  169,  281,  285,  286,  289,  290, 

336,  337,  510 
Neukirch,  536 
Neumann,  258,  400,  440 
Neustaedter,  558 
Nichols,  515 
Nicolaier,  472 
Nicolas,  438 
Nicolaysen,  303 

Nicolle,  48,  59,  466,  560,  570,  577 
Nobbe",  617 
Nocard,  347,  534,  563 
Nocht,  513 
Noguchi,  31,  157,  161,  162,  514,  515,  516, 

517,  520,  521,  523,  524,  525,  526,  529, 

558,  559,  566,  570,  574,  582 
Norris,  525 
Novy,  523,  524,  525 
Nuttall,  149,  150,  153,  488,  589 


OBERMEIER,  523 
O'Brien,  347 
Ogata,  315 
Ogsten,  270 
OhnD,  249 
Opie,  130,  440 
Orth,  261 
Osborne,  135,  141 
Osgood,  557 
Otto,  133,  262,  413 
Overbeck,  409 


PALADIN  O-BLANDINI,  63 

Paltauf,  365 

Pansini,  288,  289 

Papasotirin,  607 

Pappenheimer,  525 

Park,  392,  399,  439,  459,  474,  476,  609 

Pasquale,  513 


Pasteur,  18,  19,  256,  269,  282,  378,  493, 

565,  567 
Pastia,  558 
Paterson,  453,  558 
Patterson,  558 
Paulet,  527 
Payne,  274 
Peabody,  284,  285 
Pearce,  135 
Peckham,  328 
Perkins,  271,364,  548 
Pernossi,  476,  477 
Petersen,  138,  440 
Petresco,  518 
Petri,  471 

Petruschky,  275,  280,  313 
Pfaundler,  143 
Pfeiffer,  136,  150,  153,  308,  414,  417, 

418,  504,  509,  513 
Pfuhl,  318,  361,  419 
Philipowicz,  331 
Philipp,  274 
Pick,  142,  445 
von  Pirquet,  140 
Pittfield,  182 
Pizzini,  481 
Plant,  545 
Plaut,  530 
Plotz,  560 
Poehl,  503 
Pohl-Pincus,  444 
Polk,  533 
Pollak,  440,  487 
Poor,  566 
Popper,  557 
Forges,  156 
Possek,  419 
Possett,  330 
Poynton,  274 
Pratt,  530 

Prescott,  603,  607,  623 
Pretori,  419 
Preyss,  417 
Pribram,  286,  502 
Priesz,  382 
Prior,  511 
Proescher,  262,  350 
Proskauer,  433,  445 
Prowazek,  528,  529,  570 
Prudden,  434,  443,  459 
Pryzgode,  144 


QUENU,  480 


R 


RABINOWITSCH,  468,  471,  481 
Rahe,  385,  596,  597 
Ramonowitsch,  267 
Rankin,  391 
Ransom,  478,  479,  504 


AUTHOR  INDEX 


633 


Rattoni,  472 

Ravant,  320 

Ravenel,  438 

Raybaud,  435 

Reagh,  144 

Reed,  561 

Reichenbach,  24 

Reichert,  178 

Reinke,  65 

Rekowski,  61 

Remlinger,  566 

Reners,  506 

Renon,  362 

Reschad,  464 

Rettger,  62,  590 

Reudiger,  130 

Reuter,  518 

Richards,  321 

Richardson,  329,  336,  337 

Rickards,  209 

Ricketts,  552,  560,  561,  576 

Rideal,  248 

Rimpau,  130,  169 

Ritchie,  308 

Roddy,  350 

Rogers,  303 

Rohmann,  142 

Roland,  286,  328 

Romberg,  453 

Rosenau,  132,  394,  573 

Rosenbach,  256,  270 

Rosenow,  181,  274,  275,  281,  283,  286, 

287,  289,  451 
Rosenthal,  321 
Ross,  526 
Rost,  464 
Rotch,  597 
Rouget,  129,  482 
Roux,  374,  388,  400,  480,  504,  517,  563, 

565 

Rubner,  25 
von  Ruck,  63,  452 
Rucker,  411 
Ruppel,  58,  63,  64 
Russell,  327 


S 


SABOURAUD,  544 

Salge,  386 

Salimbini,  504 

Salmon,  344 

Sanchez,  481 

Sanfelice,  551,  554 

Sawyer,  558 

Schaeffer,  183 

Schattenfroh,  488,  498 

Schaudinn,  25,  28,  514,  515,  521,  528 

Scheffer,  58 

Schellack,  523,  525 

Schenck,  548 

Scheplewsky,  486 

Schereschewsky,  516 


Schick,  140,  399 

Schlagenhaufer,  303,  304 

Schmanowsky,  137 

Schmitt,  468 

Schmorl,  436 

Schnitzler,  361 

Schoenlein,  544 

Schottelius,  507,  589 

Schottmiiller,  271,  344,  401 

Schroeder,  19,  384 

Schultz,  135,  136 

Schiitz,  367 

Schwann,  18 

Schwarz,  472 

de  Schweinitz,  430,  436 

Sederl,  308 

Sedgwick,  384 

Seifert,  307 

Serafini,  373 

Serota,  399 

Shaffer,  598,  599 

Shattuck,  307 

Shibayama,  348 

Shiga,  315,  320,  322,  323 

Siedentoff,  178 

Silberschmidt,  361,  616 

Simmonelli,  518 

Simonds,  210,  328,  488,  491,  492,  598 

Sittler,  267 

Slawyk,  419 

vanSlyke,  218 

Smith,  R.,  492,  599 

Smith,  Theobald,  iii,  62,  70,  82,  86,  108, 
109,  115,  126,  133,  144,  170,  173,  200, 
213,  218,  220,  222,  240,  243,  272,  275, 
276,  282,  318,  344,  354,  355,  358,  360, 
361,  362,  384,  392,  393,  398,  401,  432, 
434,  435,  439,  443,  444,  458,  460,  473, 
475,  483,  491,  572,  573,  606,  623 

Smith,  W.  H.,  186,  233 

Sobernheim,  378 

Sorensen,  218 

Sormani,  481 

le  Sourd,  425 

Southard,  133,  321 

Sowade,  515,  518 

Spengler,  432 

Spiegelberg,  616 

Stefansky,  468 

Steinhardt,  134,  566 

Stern,  482 

Sternberg,  282 

Stewart,  456 

Sticker,  414,  463 

Stiegell,  25 

Stimpson,  453 

Stober,  553 

Stockmann,  384 

Straus,  371,  459 

Streit,  259 

Strong,  320 

Stuppuhn,  77,  356 

Sullivan,  53 

Surmont,  374 

von  Szekely,  375,  495 


634 


AUTHOR  INDEX 


TAKAKI,  478 
Tamura,  61 
Tarbel,  469 
Tarozzi,  482 
Tartowsky,  553 
Taurelli-Salimbini,  504 
Tavel,  278,  587 
Taylor,  361 
Tedesco,  418 
Teissier,  577 
Thiercelin,  265,  267 
Thierfelder,  589 
Thomas,  496 
Thro,  558 
Tiffeneau,  478 
Tissier,  582 
Tizzoni,  473 

Todd,  319,  320,  322,  526 
Tokishige,  553 
Toledo,  481 
Tomasczewski,  426 
Torrey,  305,  306,  386,  599 
Trask,  609 
Treitel,  419 
Trudeau,  453,  457 
Tsiklinsky,  583 
Tunnicliff,  530,  531 
Tyndall,  18,  19 
Tyzzer,  572 


UKKE,  493 


VAILLARD,  129,  322,  482 

Vallee,  498 

Vaughan,  52,  63,  76,  136,  137,  139,  435, 

448 

Veillon,  488 

van  de  Velde,  63,  130,  166,  259,  277 
Vincent,  129,  270,  482,  530,  616 
Voges,  506 
Vogt,  362 


W 


WADSWORTH,  285,  288 

Waldmann,  297 

Walker,  58,  62,  70,  78,  217,  218,  223,  248, 
257,  310,  314,  318,  326,  328,  344,  354, 
355,  358,  360,  361,  362,  364,  366,  380, 
391,  392,  433,  435,  491,  504,  599,  608, 
615,  616 

Walpole,  354 

Walsh,  577 

Warden,  303 

Warrington,  617 

Washbourn,  286 


Wassermann,    138,    142,    297,    303,    381, 

478,  486 
Webb,  457 
Weber,  438 
Wechsberg,  258 
Weeks,  423 

Weichselbaum,  282,  292,  363 
Weigert,  19,  120 
Weil,  135 

Welch,  125,  180,  264,  283,  480,  488 
Weleminsky,  62,  435 
Wells,  129,  132,  135,  141,  258,  319,  328, 

435 

Welsh,  150 
Wernstedt,  558 
Wesbrook,  389 
Wesenberg,  361 
Westenhoffer,  296 
Wheeler,  63 

Wherry,  58,  411,  412,  430,  433,  468,  555 
White,  133,  435,  444,  448 
Whittemore,  307 
Widal,  143,  340 
Wiener,  440,  506 
Wilder,  560,  561 
Wilhelmi,  267 
Willforth,  617 

Williams,  392,  457,  566,  567,  570 
Wilson,  389,  573,  576 
Winogradsky,  617 
Winslow,  623,  625 
Winternitz,  435,  441 
Woithe,  28 

Wolbach,  430,  440,  555 
Wolff,  307,  479 
Wolff-Eisner,  446,  448,  452 
Wollstein,  288,  301,  421,  422,  423 
Wood,  284 
Wright,  130,  166,  171,  184,  186,  210,  212, 

292,  293,  294,  295,  533,  536,  527,  538, 

540 
Wyssokowitsch,  261,  411 


YATES,  405,  406 
Yersin,  388,  408 
Yost,  44 


Z 


ZABOLOTNY,  411 
Zeidler,  510 
Zeit,  45,  327,  367 
Zettnow,  27,  359,  409,  523 
Zibell,  481 
Ziehl,  184,  431 
Zingher,  399 
Zinsser,  324,  440 
Zlatogoroff,  503,  511 
Zsigmondy,  178 
Zuber,  488 
Zupnik,  479 


GENERAL  INDEX. 


A 


ABDERHALDEN   theory   of    anaphylaxis, 

137 

Abortin,  383 

Abortion,  infectious,  382,  460 
Abscess  producing  cocci,  255 
Absorption  methods,  146 
Acetone-insoluble  antigen  of    Noguchi, 

157 

Achorion  schoenleinii,  19,  544,  546 
Acid  broth,  204 

formation  by  bacteria,  from  carbo- 
hydrates, 76 
from  proteins,  73 
Acid-fast  bacteria.  428-471 

distribution  of,  106,  428 
staining  methods  of,  184,  428 
Acidophilic  bacteria,  102,  204,  385 
Aciduric  bacteria,  102,  204,  385 
Acquired  immunity,  113 
Actinomyces,  106,  533,  536-541 

bovis,  536 

classification  of,  533 

cultivation  of,  538 

madursR,  541 

morphology  of,  537 

pathogen  esis  of,  539 
Active  immunity,  113 
Acute  anterior  poliomyelitis,  557 
contagious  conjunctivitis,  423 
Aerobic  bacteria,  40 
Aerobiosis,  40 
Agar,  blood,  202 

clarification  of,  191 

filtration  of,  192 

glycerin,  200 

lactose-litmus,  202 

meat  extract,  200 
infusion,  199 

oleate,  204 

preparation  of,  192,  199,  200 

reaction  of,  191 

sterilization  of,  193 
Agglutination  reaction,  143-149 
technique  of,  148,  149 
Agglutinin,  chemistry  of,  145 

flagella,  144 

group,  144,  166 

properties  of,  146 

somatic,  144 

specificity  of,  123,  143,  146,  147 

thread  reaction  of,  143 


Agglutinoid,  124,  143 
Aggressin,  165,  166 
Agitation,  effect  of,  on  bacteria,  48 
Air,  bacteria  of,  624,  625 

borne  infection,  91,  92 
Alcohol  as  disinfectant,  244 
Alcoholic  fermentation,  77 
Alexin,  151,  152 
Allantiasis,  484 
Allergy,  132-141 

Alternating  current,   effect  of,  on  bac- 
teria, 46 
Amboceptor,  121,  124,  125,  152 

multiplicity  of,  153 
Amines,  formation  of,  by  bacteria,  73, 

75 
Amino  acids  in  bacteria,  63 

utilization  by  bacteria,  73 
Ammonia,  formation  of,  by  bacteria,  73, 

80 

Amylase,  51 
Anaerobic  bacteria,  40 

cultivation  of,  209-214,  515 
distribution  of,  106 
isolation  of,  209-212 
Anaerobiosis,  40 
Anaphylactic  shock,  139-149 
Anaphylactin,  136 
Anaphylactogen,  132 
Anaphylatoxin,  135,  136 
Anaphylaxis,  132-141 
in  man,  138 
passive,  136 
theories  of,  136 
Angina,  Plaut,  530 
ulcerosa,  530 
Vincent's,  530 
Anilin  dyes,  178 

oil   as  mordant,  183 
Animals,  care  of,  240 

carriers  of  infection,  94 
inoculation  of,  237r240 
use  of,  for  diagnosis,  237 
Antagonism,  bacteria,  53,  55 
Anterior  poliomyelitis,  557 
Anthrax,  372 

bacillus,  90,  93,  98,  106,  107,  372 
asporeless,  374 
dissemination  and  prophylaxis, 

of,  90,  93,  98,  106,  107,  379 
identification  of,  378,  379 
cultural,  379 
morphological,  378 


636 


INDEX 


Anthrax    bacillus,    immunity   and    im- 
munization of ,  377-378 
isolation  and  culture  of,  374 
morphology  of,  372-374 
patbogenesis  of,  376-377 
animal,  376-377 
human,  377 

products  of  growth  of,  376 
enzymes,  376 
toxins,  376 

spores  of,  373,  374,  375,  379 
vaccines  of,  378 
intestinal,  377 
pneumonic,  377 
symptomatic,  496 
vaccine,  498 

Anthropoid   apes,   blood   serum   distin- 
guished from  human,  149 
Antianaphylaxis,  134 
Antibiosis,  bacterial,  54 
Antibodies,  nature  of,  142 
Anticomplementary  action,  158 
Antienzymes,  52 
Antiformin,  453 
Antigen,  bacterial,  163-165 
Besredka,  164 
glanders,  164 
nature  of,  142 
NogUchi,  157 

standardization  of,  157-159 
syphilitic,  156 

Antimeningococcus  serum,  297 
Antipneumococcus  serum,  290 
Antiseptics,  244 
Antistreptococcus  sera,  277 
Antitoxin,  botulinus,  486 
diphtheria,  395-398,  481 

unit,  397,  181 
tetanus,  479-484 

unit,  481 

Arnold  sterilizer,  193 
Aromatic  products  of  protein  decomposi- 
tion, 73-76 
Arthrospore,  30,  270 
Arthus  phenomenon,  140,  569 
Ascitic  fluid  media,  203 
Ash  of  bacteria,  59,  60 
Ascospores,  543,  550 
Asiatic  cholera,  499 
Aspergillus,  bouffardi,  541 
distribution  of,  235,  542 
fumigatus,  547 
mycoses,  547 
Autoclave,  193,  196 
Autogenous  vaccines,  166,  172 
Avian  tubercle  bacillus,  461,  462 
Azobacteria,  617 


B 


BABES-ERNST  granules,  27,  390 
Bacillacese,  33 
Bacillary  dysentery,  315 
Bacilli,  acid-fast,  428-471 


Bacillus,  22 

abortus,  382-385,  460 
abortin,  383 
dissemination  and  prophylaxis, 

of,  385 

identification  of,  384,  385 
cultural,  384 
serological,  384,  385 
immunity  and  immunization  of, 

383,  384 

isolation  and  culture  of,  382 
morphology  of,  382 
pathogenesis  of,  383 
products  of  growth  of,  383 
acidophilus,  104,  107,  385,  386 

dissemination  of,  104,  107,  385 
isolation  and  culture  of,  386 
morphology  of,  385,  386 
pathogenesis  of,  386 
types  of,  385 
aerogenes    capsulatus,    90,    93,    98, 

104,  106,  107,  488-493 
dissemination  and  prophyl- 
axis  of,  90,  93,  98,  104, 
106,  107,  493 
isolation    and    culture    of, 

489-491 

morphology  of,  488,  489 
pathogenesis  of,  492,  493 
products  of  growth  of,  491 
enzymes,  491 
hemolysin,  489 
toxin,  491 
types  of,  492 
Welch-Nut  tall  test,  490 
aertrycke,  348 
alcaligenes,  313-315 

ammonia  formation,  80 
dissemination  and  prophylaxis 

of,  107,  315 

identification  of,  314,  315 
immunity  of,  314 
isolation  and  culture  of,   313, 

314,  316 

morphology  of,  313 
pathogenesis  of,  314 
products  of  growth  of,  80,  222, 

314,  316 
anthracis,  372-379 

symptomatic*,  93,  98,  496-498 
dissemination  of,  93,  98 
immunity  and  immuniza- 
tion of,  498 
isolation    and    culture    of, 

496,  497 

morphology  of,  496 
pathogenesis  of,  498 
products  of  growth  of,  497 
enzymes,  497 
toxin,  498 
vaccine  of,  498 
avisepticus,  407 
bifidus,  103,  104,  107,  582 
of  Bordet  and  Gengou,  420-423 
bottle  of  Melassez,  106 


INDEX 


637 


Bacillus  botulinus,  52,  90,  94,  102,  107, 

344,  484-488 
antitoxin,  486 
dissemination  and  prophylaxis 

of,  94,  107,  344,  488 
identification  of,  487 
cultural,  487 
microscopic,  487 
by  toxin,  487 
immunity  and  immunization  of, 

486-488 

isolation  and  culture  of,  484 
morphology  of,  484 
pathogenesis  of,  486,  487 
products  of  growth  of,  485,  486 
toxin,    52,    102,    485, 

486,  487 

bulgaricus,  385,  596 
butter,  471 

isolation  and  culture  of,  471 
morphology  of,'  471 
pathogenesis  of,  471 
chlorimum,  27 

chlorophyll  in,  27 
cholerse  suis,  349 
cloacaB,  80,  107,  316,  358 
ammonia  formation,  80 
distribution  of,  107,  316 
isolation  and  culture  of,  358 
morphology  of.  358 
products  of  growth  of,  358 
clostridium  pasteurianum,  617 
coli,  74,  80,  82,  102,  104-106,  218, 

222,  316,  353-357.  621 
ammonia  formation,  80 
dissemination  of,  102,  104-107, 

356 

identification  of,  357,  621 
immunity  and  immunization  of, 

357 
isolation  and  culture  of,   203, 

353,  354 

morphology  of,  353 
pathogenesis  of,  356,  357 
products  of  growth  of,  74,  82, 

203,  218,  354,  355 
enzymes,  355,  356 
indol,  74,  82,  218,  356 
milk,    203,    222,    316, 

356 

toxins,  356 
in  water,  621 
of  Danyz,  348 
definition  of,  33 

.  diphtherias,  388-404.     See  Diphthe- 
ria bacillus, 
of  Doderlein,  104 
of  Ducrey,  42^427 

identification  of,  427 

by  autoinoculation,  427 
cultural,  427 
microscopic,  427 
isolation  and  culture  of,  426 
morphology  of,  426 
pathogenesis  of,  426,  427 


Bacillus     dysenteriae,      315-324.        See 
Dysentery  bacillus. 

Flexner,  315-324 

Hiss-Russell,  316 

Rosen,  316 

Shiga,  315-324 
enteritidis,  344,  347,  348 
of  Friedlander,  363 
of  Fraenkel,  353 
fusifprmis,  106,  530,  531 

dissemination  of,  106 
-—isolation  and  culture  of,  530 

morphology  of,  530 

pathogenesis  of,  531 
of  Gartner,  347 
geniculatus,  102 
of  glanders,  367-372.     See  Glanders 

bacillus, 
grass,  470,  471 

of  hemorrhagic  septicemia,  407-416 
of  Hofmann,  404 

toxin,  404 
hodgkini,  405,  406 

isolation  and  culture  of,  405 

morphology  of,  405,  406 

pathogenesis  of,  406 
icteroides,  80,  347 
influenzas,  417-420.      See  Influenza 

bacillus. 

of  Karlinski,  467,  470 
of  Kedrowski,  464 
Koch- Weeks,  423-424 

dissemination  of,  106 

isolation  and  culture  of,  424 

morphology  of,  423,  424 

pathogenesis  of,  424 

products  of  growth  of,  424 
Kopfchen,  581 
lactis  aerogenes,  107,  366 

viscosus,  608 

leprse,  463-468.    See  Leprosy  bacil- 
lus. 

rat  leprosy,  468 
of  Lustgarten,  455 
mallei,  164,  367-372.    See  Glanders 

bacillus, 
melitensis.    See  Micrococcus   meli- 

tensis. 
mesentericus,  104,  107,  222,  615 

distribution  of,  104,  107,  615 

reaction  of,  in  milk,  222,  223 
Morax-Axenfeld,  106,  424,  425 

dissemination  of,  106 

isolation  and  culture  of,  425 

pathogenesis  of,  425 

products  of  growth  of,  425 
of  Morgan,  80,  221 
moorseele,  347 
morbificans  bovis,  347 
mucosus  capsulatus,  363 

isolation  and  culture  of,  364 

morphology  of,  363 

pathogenesis  of,  365 
neapolitanus,  353 
cedematis'maligni,  493-496 


638 


INDEX 


Bacillus    oedematis  maligni,   dissemina- 
tion and  prophylaxis  of, 
93,  98,  493,  496 
immunity   and   immuniza- 
tion of,  496 
isolation    and    culture    of, 

494,  495 

morphology  of,  493,  494 
pathogenesis  of,  495,  496 
products  of  growth  of,  495 
enzymes,     495 
toxins,  495 
ozenae,  106,  236,  365 
paratyphosus  alpha  and  beta,  344- 

352 
ammonia      formation, 

80 

carriers  of,  351 
dissemination   of,    94, 

104,  107,  349,  353 
fermentation  reactions 

of,  221,  316 
identification  of,  350 
cultural,  350 
serological,  351 
immunity  and  immu- 
nization of,  352 
isolation    and    culture 

of,  345,  350 
morphology  of,  345 
pathogenesis  of,  348 
meat     poisoning, 

348-350 

products  of  growth  of, 
203,     222, 
316,  346 
chemical, 
203,      222, 
316 

enzymes,  346 
toxins,     102, 

^  347,  350 

synonyms  of,  344,  345 
perfringens,  488 
pertussis,  420-423 

dissemination  of,  93,  106,  421 
identification  of,  423 
cultural,  423 
microscopic,  423 
serological,  423 
immunity  of,  422 
isolation  and  culture  of,  421 
morphology  of,  420 
pathogenesis  of,  422 
animal,  422 
human,  422 
products  of  growth  of,  421,  422 

toxins,  421,  422 
pestis,  93,  95,  106,  107,  110,  407- 

416.    See  Plague  bacillus, 
phlei,  470-471 

dissemination  of,  469 
isolation  and  culture  of,  470 
morphology  of,  470 
pathogenesis  of,  471 


Bacillus  phlei,  synonyms  of,  470 

of   plague,    407-416.       See    Plague 

bacillus. 

in  rodents,  413-416 
pneumobacillus,  106,  363 
pneumonia?,  363 
propionic  acid,  353 
proteus  group,  316,  359 
fluorescens,  362 
vulgaris,  359-362 

dissemination  of,  104, 

107,  362 
isolation    and    culture 

of,  359,  360 
morphology  of,  359 
pathogenesis  of,  361 
products  of  growth  of, 

360,  361 
enzymes,  81, 

361 
indol,  78,  82, 

218,  361 
toxins,  361 
zenkeri,  359 
zopfii,  359 

pseudodiphtheria?,  391,  404-406 
pseudotuberculosis  rodentium,  413, 

416 
psittacosis,  106,  347,  351,  352 

dissemination    of,      106,     347, 

348 

identification  of,  352 
pathogenesis  of,  351,  352 
putrificus,  581 
pyocyaneus,  379-382 

ammonia  formation,  80,  106 
identification  of,  382 
immunity  and  immunization  of, 

382 

isolation  and  culture  of,  380 
morphology  of,  379,  380 
pathogenesis  of,  381 
animal,  381 
human,  381 

products  of  growth,  380,  381 
chemical,  380,  381 
enzymes,  381 
pigments,  380,  381 
pyocyanin,  380,  381 
toxins,  381 

pyogenes  fcetidus,  316 
radicicola,  617 
of  rat  leprosy,  468 

plague,  347 

rhinoscleromatis,  106,  236,  365 
smegmatis,  98,  105,  106,  455,  469 
dissemination  of,  98,  105,  106, 

455  . 

morphology  of,  469,  470 
pathogenesis  of,  470 
__of  soft  chancre,  425-427 
subtilis,  107,  615-617 
suipestifer,  347 
suisepticus,  347 
swine  plague,  347 


INDEX 


639 


Bacillus  tetani,  472-484.     See  Tetanus 

bacillus. 

tuberculosis,  429-462 
avian,  461,  462 
bovine,  457-460 
human,  429-457.    -See  Tubercle 

bacillus. 

ichthic  type,  429 
tularense,  412,  416 
typhi  murium,  348 
typhosus,    325-343.      See    Typhoid 

bacillus, 
viride,  26 

chlorophyll  in,  26 
xerosis,  99,  404,  405 
welchii,     488-493.       See     Bacillus 

aerogenes  capsulatus. 
Bacteria,  anaerobic,  472-498 
as  antigens,  163-165 
branching  of,  24 
chemistry  of,  56-67 
chromogenic,  53,  380 
cultivation  of,  187-223 
counting  of,  206,  207,  215-217.    See 

also  under  Milk  and  Water, 
deaminization  by,  73,  79,  80,  218 
definition  of,  17 
degeneration  of,  23 
destruction  of.    See  Sterilization, 
distribution  of,  general,  17 

parasitic  and  pathogenic,  89,  90 
enzymes  of,  49-53 
examination  of,  in  living,  176-178 
function  of,  in  nature,  17,  18,  56 
growth  of,  in  animal  body,  55,  105 
isolation  of,  206-214 
morphology  of,  abnormal  forms,  23 

normal  forms, -21 
media  for,  189,  204.    See  also  under 

Specific  organisms, 
metabolism  of,  68-83 
nitrogen  cycle  of,  17,  18,  57,  617-620 
as  opportunists,  87,  225,  274 
parasitic,  18 
pathogenic,  18,  255-532 
relation  of,  to  plants  and  animals,  17 
saprophytic,  18 
staining  of,  178-187 
stains  for,  178-187 
toxins,  49-53 
.r-    3ines,  166-174 
Bach      il  suspensions  for  opsonic  index 

des     minations,  167,  168 
Bacteriology,  definition  of,  17 

historical,  18-20 
Bacteriolysin,  51 
Bacteriopurpurin,  52 
Bacteriotropins,  130,  166-174 

nature  of,  169 
Bacterium,  definition  of,  33 
Bail  aggressin  theory,  165,  166 
Balanced  pathogenism,  107 
Barber  single  cell  isolation  method,  208 
Bath  water,  sterilization  of,  253 
Beggiatoa,  106 


Berkefeld  filters,  194,  195 
Betaimidazoleethylamine,  76 
Black  leg,  496 

vaccine,  498 

Bladder,  bacteria  of,  105 
Blastomycetes,  106,  551-554 
Bleach  as  germicide,  245 
Blindschleiche  bacillus,  469 
Blood  agar,  202 

bacteria  in,  107 

cultures,  technic  of,  225 

serum,  Loffler's,  200 
Blue  pus,  379 

Bordet-Gengou  bacillus,  420 
Boric  acid  as  germicide,  247 
Botulism,  484 
Bouillon,  acid,  204 

ammonia  formation  in,  80,  218 

calcium  carbonate,  198 

chemical    changes    in,    induced    by 

bacteria,  217-221 
composition  of,  217-218 

clarification  of,  191 

deaminization  in,  80,  218 

dextrose,  197 

Dunham,  198 

filtration  of,  192 

glycerin,  198 

growth  of  bacteria  in,  212-214,  217 

lactose,  197 

mannite,  197 

meat  extract,  197 

infusion,  196 
sugar-free,  197,  218 

nitrate,  199 

preparation  of,  196 

reaction  of,  191,  204 

saccharose,  197 

sterilization  of,  193 

sugar,  197 

sugar-free,  197,  218 
Bovine  tubercle  bacillus,  438,  457-461 
Branching  in  bacteria,  24 
Brill's  disease,  559 
Bromatherapy,  597-600 
Brownian  movement,  28 
Bubonic  plague,  407-416 
Buccal  material,  bacteria  in,  106,  232 
examination  of,  232 


CADAVERIN,  75 

Calcium  carbonate  broth,  198 

Calmette  ophthalmo- tuberculin  reaction, 

450 

Cancer  and  yeasts,  551 
Capsule,  bacterial,  26 

chemical  composition  of,  62,  82 

stains,  180 
Carbohydrates  in  bacterial  cell,  64 

decomposition  of,  by  bacteria,   77, 
219-221 

enzymes,  splitting,  51 


640 


INDEX 


Carbohydrates  as  food  for  bacteria,  66 
influence  of,  on  bacterial   metabo- 
lism, 76,  80-83,  218-221 
media,  197,  219 
Carbol  fuchsin,  180 
Gar.bolic  acid  coefficient,  248 
as  disinfectant,  246 
Carbon  metabolism  of  bacteria,  72 

sources  for  bacteria,  66 
Carboxylase,  73,  75,  77 
Carboxylic  decomposition  by  bacteria, 

73,  75,  77 
Carriers,  animal,  94 

cholera,  96,  510,  511 
dysentery,  96,  324 
human,  95 
insects,  94 
paratyphoid,  96,  351 
typhoid,  96,  330,  331 
Cell  division  in  bacteria,  31 
grouping  in  bacteria,  32 
membrane  of  bacteria,  25 

chemical    composition   of, 

61 

receptors,  119-126 
substance  of  bacteria,  26 
Cellular  elements  in  milk,  613 

theory  of  immunity,  117-131 
Cellulase,  51 

Cellulose  in  bacteria,  61,  62 
Cerebrospinal  fluid,  bacteria  in,  106 
cultures  of,  226-228 

technique  of,  226 
meningitis,  292 
Chancroid,  425 
Charbon  symptomatique,  496 
Chemical  composition  of  bacteria,  58,  59, 

80,82 

constitution  of  bacteria,  58 
Chemistry  of  bacteria,  56-77 
Chemotaxis,  129 

influence  of,  on  bacteria,  49 
Chitin  in  bacteria,  62 
Chlamydospore,  545,  547 
Chlorinated  lime,  245 
Chlorine  as  germicide,  245,  251,  252 
Chlorophyll  in  bacteria,  26,  27 
Cholera  asiaticae,  499 

group  of  vibrios,  499,  511,  513 

nostras,  511 

red  reaction,  499,  503 

sicca,  506 

vibrio,  4997511 

agglutination  of,  510 
ammonia  formation,  80 
carriers  of,  96,  510,  511 
dissemination  and  prophylaxis 
of,  93,  94,  96,  104,  107,  510 
identification  of,  507-511 
agglutination,  510 
complement  fixation,  510 
cultural,  507,  508 
microscopical,  507 
Pfeiffer's  phenomenon,  509 
serological,  508-510 


Cholera  vibrio,  immunity  and  immuni- 
zation of,  507 

isok  tion  and  culture  of,  501 
morphology  of,  499-501 
pathogenesis  of,  506 
animal  505 
human,  506 

products  of  growth  of,  82,  503 
cholera  red,  503 
enzymes,  82 
hemolysin,  502,  503 
toxins,  504,  505 
Chromogenic  bacteria,  53,  380 
Cladothrix,  33,  533,  534 
Clarification  of  media,  191 
Clostridium  pasteurianum,  617 
Coagulase,  51 
Cocci,  pyogenic,  255-268 
Coccus,  21,  33 

Cold,  effect  of,  on  bacteria,  42 
Colony  enumeration,  215-217.    See  also 

Milk  and  Water, 
formation,  214-217 
Complement,  153 
fixation,  154 

technique  of,  156-165 
multiplicity  of,  153 
preparation  of,  159 
Conjunctiva,  bacteria  of,  98 
Conjunctivitis,  acute,  234,  423 
contagious,  423 
pseudomembranous,  235 
subacute,  235,  424 
Contact  infection,  96 
Contagious  pleuropneumonia  of  cattle, 

563 
Continuous  electric  current,  effect  of,  on 

bacteria,  45 

Corrosive  sublimate  as  germicide,  244 
Crenothrix,  33 
Cresols,  effect  of,  on  bacteria,  246 

produced  by  bacteria,  75 
Cryptogenetic  tetanus,  482 
Cultures  of  bacteria,  incubation,  214 
methods,  213-223,  473,  496 
solid  media  for,  214-217 
Cutaneous  tuberculin  test,  449 
Cycle  of  nitrogen,  17,  18,  56,  57,  617-620 
of  parasitism,  86,  87 
of  pathogenism,  87-89 ' 
Cytolysins,  125 
Cytoplasm  of  bacteria,  26 

chemical  composition  of,  62 
Cytoryctes  variolse,  571 


DARK  field  illumination,  178 
Deaminization  by  bacteria,  73,  7»,  80, 

218 

Defenses  of  body  against  infection,  97 
Degeneration  in  bacteria,  23 
Dengue,  576 
Dental  instruments,  sterilization  of,  254 


INDEX 


641 


'^T~/  effect  oi^.^n .bacteria,  39 

^14-254.     See 
on. 
tuberculin 

398 

concentration  of,  396 
nmitive  value  of,  398 
397 

it  ion  of,  397-398 
unit  of,  397 
bacillus,  388-404 

ammonia  formation,  80 
cellulose  in,  61 

dissemination  and  prophylaxis 
of,  90,  93,  100,  101, 
106,  403,  404 
use  of  antitoxin,  403 
serum  sickness,  403 
identification  of,  401-403 
microscopical,  401,  402 
toxin  formation,  402,  403 
immunity    and    immunization, 

398,  399,  403 
Schick  reaction,  399 
with    toxin    antitoxin 

mixtures,  398 
isolation  and  culture  of,  390- 

392,  405 

morphology  of,  388,  389 
pathogenesis  of,  400,  401 
animal,  398,  400 
human,  400,  401 
products  of  growth  of,  392,  403 
chemical,  392 
enzymes,  392 
toxin,  52,  82,  102,  109, 

218,  392,  403 
action,  395 
constitution,  394 
production,  392 
storage,  393 
testing     potency, 

393,  394 
toxoid,  395 
toxone,  395 
prophylactic  immunization  of, 

403 
group,  388-406 

toxin,   52,   82,    103,    109,   218, 

392-395,  480 

Diplococcus  catarrhalis,  307-309 
dissemination  of,  106 
identification  of,  308,  309 
isolation  and  culture  of,  308 
morphology  of,  307,  308 
pathogenesis  of,  308 
products  of  growth  of,  308 
definition  of,  33 

}.    norrhoeae,  301.    See  Gonococcus. 
intraccllularis  meningitidis,  292.  See 

Meningococcus. 
lanceolatus,  282-291.    See  Pneumo- 

coccus. 

pneumonia?,  282-291 
weiohselbaumii,  292 
41 


Discomyces  bovis,  536 
Disinfectants,  244-248 

chemical  solutions,  244 
gaseous,  249-253.    See  Sterilization. 
Dohle  bodies  in  scarlet  fever,  564 
Double  sugar  media,  204 
Droplet  infection,  92 
Dry  heat,  effect  of,  on  bacteria,  43 
Drying,  effect  of,  on  bacteria,  39,  40 
Ducrey  bacillus,  425 
Dunham  solution,  198 
Dust  infection,  91 
Dysentery  bacillus,  315-324 

Flexner  and  Shiga  types,  315 
ammonia  formation,  80 
dissemination  and  prophyl- 
axis of,  93,  96, 104,  107, 
324 

identification  of,  323 
immunity  and  immuniza- 
tion of,  322 
isolation    and    culture  of, 

317,  318 

morphology  of,  316, 
pathogenesis  of,  320 
products  of  growth  of,  316, 

318,  221,  222 
chemical,  316, 318, 

321 
toxins,  318 


E 


EAR,  bacteria  in,  106,  235,  362,  365 
Ectoplasm  of  bacteria,  25 

chemical  composition,  58 
Edema,  malignant,  493-496 
Egg  media,.  203 

Ehrlich  theory  of  immunity,  117-126 
Einheit  of  streptococci,  280 
Electricity,  effect  of,  on  bacteria,  44-46 
Emphysematous  gangrene,  bacteria  of, 

488^493 
Emulsin,  51 

Endo  medium,  201,  330 
Endospores,  29 
Endotoxins,  52 
Engulf ment  of  bacteria  by  leukocytes?- 

1297131 

Enteritidis  group,  344 
Enteritis,  streptococcus,  275 
Enterococcus,  265-268.     See  Micrococ- 

cus  ovalis. 
Enzymes,  bacterial,  49 

classification  of,  50 

endo-,  50 

exo-,  49,  78,  217 

general,  50 

influence  of  carbohydrates  on, 
78,  217 

properties  of,  52 
Epidemic  cerebrospinal  meningitis,  292 

poliomyelitis,  557 
Epidemiology,  107 


642 


INDEX 


Ernst-Babes  granules,  27,  390 
Erythrocytes    for   complement   fixation 

test,  159 

Erythrocytolysis.    See  Hemolysis. 
Essential  oils,  germicidal  action  of,  248 
Esterase,  51.     See  Specific  organisms. 
Eubacteriacese,  33 
Eumycetes,  541 
Eury  thermic  bacteria,  42 
Examination  of  air,  624,  625 

of  milk,  601-613 

of  various  organs  and  tissues  of  body, 
224-237 

of  water,  618-623 
Eye,  bacteria  of,  106,  234 


FARCIN,  534 

Farcy,  367 

Fat  in  bacteria,  64 

enzymes,  splitting,  51 

in  tubercle  bacillus,  64,  65 
Favus,  543 
Feces,  bacteria  in,  107,  2,30^232,  579-600 

sterilization  of,  252,  253 
Fermentation  by  bacteria,  83 

enzymes  of,  51 

tubes,  213-219,  220,  473,  496 
Film  preparations,  178-187 
Filterable  viruses,  178,  555 
Filters,  bacterial,  194,  556 

Berkefeld  and  Chamberland,    194, 
195,  556 

care  of,  556 

testing,  556 

water  and  sewage,  619,  620 
Filtration  of  media,  192 
Fixation  of  complement,  154 

technique  of,  156-165 
Flagella,  28 

stains  for,  182 

Flies  as  carriers  of  infection,  94 
Fluorescent  bacteria,  53 
Fomites,  disinfection  of,  253 
Food  borne  infections,  93 

relations  of  bacteria  to,  65 

sources  for  bacteria,  66 
Foot  and  mouth  disease,  562 
Formaldehyde.    See  Formalin. 
Formalin,  germicidal  properties  of,  247 
Formiase,  77,  356 
Fraenkel-Gabbet  stain,  184 
Frambesia,  526 

Freezing,  effect  of,  on  bacteria,  42 
Friedberger  theory  of  anaphylaxis,  137 
Friedlander  bacillus,  363 
Fungi,  541-554 
Fusiform  bacillus,  530-532 


G 


G/   L-STONES,  bacteria  in,  357 
Garget,  streptococci  in,  276 
Gartner  group  of  bacteria,  344 


Gas  bacillus,  488 

formation  of,  by  bacteria,  219,  220 
aseous  disinfection,  249-252 
astro-intestinal  bacteriology,  107,  230- 
232,  579-600 
Gelase,  51 

Gelatin,  bacterial  growth  in,  217 
clarification  of,  191 
colonies  in,  217 
composition  of,  217 
filtration  of,  192 
incubation  of,  207 
liquefaction  of,  217 
preparation  of,  199 
reaction  of,  191 
sterilization  of,  193 
Germinal  infection,  96,  467 
Germination  of  spores,  37 
Giemsa  stain,  185,  186 
Glanders  bacillus,  367-372 

dissemination  and  prophylaxis 

of,  372 

identification  of,  164,  370,  371 
complement  fixation,  164 
Straus  reaction,  371 
immunity  and  immunization  of, 

369,  370 

isolation  and  culture  of,  368 
morphology  of,  367 
pathogenesis  of,  237,  369-370 
products  of  growth  of,  368 

mallein     or     morvin, 

368,  369,.  372 

Glassware,  sterilization  of,  188-190 
Glucose  media.     See  Dextrose. 
Glucoseamine  in  bacteria,  62 
Glucoside-splitting  enzymes,  51 
Glycerin  agar,  200 
broth,  198 
egg  media,  203 
Gonococcus,  301-307 

dissemination    and  prophylaxis   of, 

99,  104-106,  307,  570 
identification  of,  305-307 

microscopical,  305,  306 
serological,  306,  307 
immunity  of,  305 
isolation  and  culture  of,  302,  303 
morphology  of,  302 
ophthalmia  neonatorum,  99,  304 
pathogenesis  of,  304,  305 
products  of  growth  of,  303,  30^ 

toxins,  303,  304 
Gram  stain,  182-184,  186,  187 

theory  of,  182,  183 
Granules  in  bacteria,  27,  390 
Ernst-Babes,  27,  390 
metachromatic,  27,  28 
polar,  27 

Grass  bacilli,  469,  470 
Gravity,  effect  of,  on  bacteria,  46 
Group  agglutinins,  146 
Growth  of  bacteria  in  animal  body,  55 
Guarnieri  bodies,  571,  572 
|  Guinea-pig  anatomy,  238 


INDEX 


651 


ULTRAMICROSCOPE,  178 
Ultramicroscopic  examination  of  bacte- 
ria,  178 

viruses,  555-564 
Urease,  51 

Ureter,  bacteria  of,  105 
Urethra,  bacteria  of,  105 
Urinary  bladder,  bacteria  of,  105,  361 
Urine,  bacteria  in,  229,  470 

collection  of,  229 

disinfection  of,  252 
Uterus,  bacteria  of,  104 


VACCINATION  against  smallpox,  574-576 
Vaccine,  bacterial,  166-174 

dosage  of,  173 

indications  for  use  of,  166-174 

preparation  of,  171-173 

prophylactic,  170 

therapeutic,  170 

therapy  of  Wright,  166-174 

virus,  572-574 

bacteria  in,  574 
Noguchi  germ-free,  574 
preparation  of,  572-574 
sources  of,  573 
tetanus  spores  in,  481,  574 
Vaccinia,  571 
Vagina,  bacteria  of,  104 
Variola,  571 

Vaughan  theory  of  anaphylaxis,  136 
Vibrio  cholerse,  499 

el  Tor,  502 

Nasik,  502 

proteus,  511 
Vibrion  septique,  493 
Vielheit  of  streptococci,  280 
Vincent's  angina,  530 
Virus  fixe,  568 
Vomitus,  disinfection  of,  252 


W 


WARM  stage  for  bacteria,  177 
Wassermann  reaction,  156-165,  520 


Water  bacteria,  352,  618 

borne  infection,  93,  620 

contamination  of,  620 

examination  of,  621 

interpretation  of  analyses  of,  622 

purification  of,  by  bacteria,  619 
and  nitrogen  cycle,  619 

standards  of  purity  of,  622-625 
Weil's  disease,  362 
Welch  bacillus,  488 

capsule  stain,  180 
Welch-Nuttall  test,  490 
Whooping-cough,  420 
Widal  reaction,  339-343 
Woolsorter's  disease,  377 
Wounds,  bacteria  of,  98 
Wright's  anaerobic  culture  method,  210, 
212 

stain,  184,  185 


XANTHIN  bases  in  bacteria,  63 
Xerosis  bacillus,  99,  404,  405 
distribution  of,  99 
morphology  of,  404,  405 
pathogenesis  of,  405 
X-r£  rs,  effect  of,  on  bacteria,  46 


YAWS,  526 

Yeasts,  102,  549.  See  also  Blastomycetes. 

Yellow  fever,  561,  562 

dissemination  of,  561,  562 

etiology  of,  561 

immunity  of,  562 

mosquitoes  in,  562 


ZIEHL-NEELSEN  stain,  184 
Zooglea,  bacterial,  26 
Zygospore,  542 
Zymase,  51,  550 


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